Cell-cycle checkpoint genes

ABSTRACT

This invention relates to a class of checkpoint genes and their polypeptide products which control progression through the cell cycle in eukaryotic cells. In particular this invention relates to  Schizosaccharomyces pombe  rad3 gene, to its human homologue (ATR) and to their encoded proteins. The invention further relates to assay methods for selecting compounds which modulate the activity of the polypeptide products of these checkpoint genes and the use of the selected compounds in anticancer therapy.

BACKGROUND OF THE INVENTION

[0001] Control of the cell cycle is fundamental to the growth andmaintenance of eukaryotic organisms, from yeasts to mammals. Eukaryoticcells have evolved control pathways, termed “checkpoints” which ensurethat individual steps of the cell cycle are completed before the nextstep occurs. In response to DNA damage, cell survival is increased bothby direct DNA repair mechanisms and by delaying progression through thecell cycle. Depending on the position of the cell within the cycle atthe time of irradiation, DNA damage in mammalian cells can prevent (a)passage from G1 into S phase, (b) progression through S phase or (c)passage from G2 into mitosis. Such checkpoints are thought to preventdeleterious events such as replication of damaged DNA and thesegregation of fragmented chromosomes during mitosis (Hartwell andKastan, 1994).

[0002] The rad3 gene of Schizosaccharomyces pombe is required for thecheckpoints that respond to DNA damage and replication blocks. Rad3 is amember of the lipid kinase subclass of kinases which possess regionshaving sequence homology to the lipid kinase domain of the p110 subunitof phosphatidylinositol-3 kinase (PI-3 kinase). This subclass alsoincludes the ATM protein defective in ataxia-telangiectasia patients.Cells from aeaxia telangiectasia patients (AT cells) have lost the delayto S phase following irradiation and are said to display radio resistantDNA synthesis (Painter and Young, 1989). AT cells irradiated in S phaseaccumulate in G2 with lethal damage, presumably as a consequence ofattempting to replicate damaged DNA. AT cells irradiated during G2display a different phenotype, they do not arrest mitosis after DNAdamage, and progress through mitosis with damaged DNA (Beamish andLavin, 1994). Mutations at the A-T locus, to which the ATM gene has beenmapped, thus result in disruption of several checkpoints required for anappropriate response to ionising radiation. Other members of this lipidkinase subclass include: Te11p (Greenwell et al. 1985). a gene involvedin maintaining proper telomere length in Saccharomyces cerevisiae Esr1p;Mec1p and the product of the Drosophila melanogaster mei-41 checkpointgene (Hari et al. 1995).

DISCLOSURE OF THE INVENTION

[0003] We have analyzed the S. pombe rad3 gene and found that it has afull length amino acid sequence of 2386 amino acids, not the 1070 aminoacids described by Seaton et al. 1992. We have determined that this isthe direct homologue of S. cerevisiae Esr1p, and that it shares the sameoverall structure as the ATM gene. The C-terminal region of the rad3protein contains a lipid kinase domain, which is required for Rad3function We have shown that Rad3 is capable of self association. We havealso identified a protein kinase activity associated with Rad3.

[0004] Further, we have found a human homologue to ram. This gene, whichwe have named ATR (ataxia and rad related), displays significantlyhigher homology to rad3 than it does to the ATM gene.

[0005] The human ATR cDNA sequence is set out as Seq. ID No. 1. Theamino acid sequence of the ORF from nucleotides 80 and 8011 is set outas Seq. ID No. 2.

[0006] The DNA sequence of the open reading frame (ORF) of rad3 is shownas Seq. ID. No. 3. The 2386 amino acid translation of the gene(nucleotides 585 to 7742 of Seq. ID No. 3) is shown as Seq. ID. No. 4.

[0007] Accordingly, in a first aspect, the invention provides the ATRprotein of Seq. ID. 2 and homologues thereof, polypeptide fragmentsthereof, as well as antibodies capable of binding the ATR protein orpolypeptide fragments thereof. ATR proteins, homologues and fragmentsthereof are referred to below as polypeptides of the invention.

[0008] In another aspect, the present invention provides apolynucleotide in substantially isolated form capable of hybridisingselectively to Seq.ID No 1 or to the complement (i.e. opposite stand)thereof. Also provided are polynucleotides encoding polypeptides of theinvention.

[0009] Such polynucleotides will be referred to as a polynucleotide ofdie invention. A polynucleotides of the invention includes DNA of Seq.IDNos 1 and fragments thereof capable of selectively hybridising to thisgene.

[0010] In a further aspect, the invention provides recombinant vectorscarrying a polynucleotide of the invention, including expressionvectors, and methods of growing such vectors in a suitable host cell,for example under conditions in which expression of a protein orpolypeptide encoded by a sequence of the invention occurs.

[0011] In an additional aspect, the invention provides kits comprisingpolynucleotides, polypeptides or antibodies of the invention and methodsof using such kits in diagnosing the presence of absence of ATR and itshomologues, or variants thereof, including deleterious ATR mutants.

[0012] The invention further provides assay methods for screeningcandidate substances for use as compounds for inhibiting or activatingATR activity, or the activity of mutated forms of ATR which aredeficient in checkpoint activity. The invention also provides assaymethods for screening candidate substances for use as compounds forinhibiting interactions between ATR and other compounds that interactwith ATR, including ATR itself.

[0013] In a related aspect, the invention also provides a polynucleotidesequence of Seq. ID No. 3 in substantially isolated form, and theprotein of Seq. ID No. 4 in substantially isolated form, and novelfragments and variants thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0014] A. Polynucleotides.

[0015] Polynucleotides of the invention may comprise DNA or RNA. Theymay also be polynucleotides which include within them synthetic ormodified nucleotides. A number of different types of modification tooligonucleotides are known in the an. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of thepresent invention, it is to be understood that the polynucleotidesdescribed herein may be modified by any method available in the art.Such modifications may be carried out in order to enhance the in vivoactivity or lifespan of polynucleotides of the invention.

[0016] Polynucleotides of the invention capable of selectivelyhybridizing to the DNA of Seq. ID No. 1 will be generally at least 70%,preferably at least 80 or 90% and more preferably at least 95%homologous to the corresponding DNA of Seq. ID No. 1 over a region of atleast 20, preferably at least 25 or 30, for instance at least 40, 60 or100 or more contiguous nucleotides.

[0017] It is to be understood that skilled persons may, using routinetechniques, make nucleotide substitutions that do not affect thepolypeptide sequence encoded by the polynucleotides of the invention toreflect the codon usage of any particular host organism in which thepolypeptides of the invention are to be expressed.

[0018] Any combination of the above mentioned degrees of homology andminimum sizes may be used to define polynucleotides of the invention,with the more stringent combinations (i.e. higher homology over longerlengths) being preferred. Thus for example a polynucleotide which is atleast 80% homologous over 25, preferably 30 nucleotides forms one aspectof the invention, as does a polynucleotide which is at least 90%homologous over 40 nucleotides.

[0019] Polynucleotides of the invention may be used to produce a primer,e.g. a PCR primer, a primer for an alternative amplification reaction, aprobe e.g. labelled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides may becloned into vectors. Such primers, probes and other fragments will be atleast 15, preferably at least 20, for example at least 25, 30 or 40nucleotides in length, and are also encompassed by the termpolynucleotides of the invention as used herein.

[0020] Polynucleotides such as a DNA polynucleotide and primersaccording to the invention may be produced recombinantly, synthetically,or by any means available to those of skill in the art. They may also becloned by standard techniques.

[0021] In general, primers will be produced by synthetic means,involving a step wise manufacture of the desired nucleic acid sequenceone nucleotide at a time. Techniques for accomplishing this usingautomated techniques are readily available in the art.

[0022] Longer polynucleotides will generally be produced usingrecombinant means, for example using a PCR (polymerase chain reaction)cloning techniques. This will involve making a pair of primers (e.g. ofabout 15-30 nucleotides) to a region of the ATR gene which it is desiredto clone, bringing the primers into contact with mRNA or cDNA obtainedfrom a human cell (e.g. a dividing cell such as a peripheral bloodleukocyte), performing a polymerase chain reaction under conditionswhich bring about amplification of the desired region, isolating theamplified fragment (e.g. by purifying the reaction mixture on an agarosegel) and recovering the amplified DNA. The primers may be designed tocontain suitable restriction enzyme recognition sites so that beamplified DNA can be cloned into a suitable cloning vector.

[0023] Such techniques may be used to obtain all or part of the ATRsequence described herein. Genomic clones containing the ATR gene andits introns and promoter regions may also be obtained in an analogousmanner, staring with genomic DNA from a human cell, e.g. a liver cell.

[0024] Although in general the techniques mentioned herein are wellknown in the art, reference may be made in particular to Sambrook et al.(Molecular Cloning: A Laboratory Manual, 1989).

[0025] Polynucleotides which are not 100% homologous to the sequences ofthe present invention but fall within the scope of the invention can beobtained in a number of ways.

[0026] Other human allelic variants of the ATR sequence described hereinmay be obtained for example by probing genomic DNA libraries made from arange of individuals, for example individuals from differentpopulations.

[0027] In addition, other animal, particularly mammalian (e.g. mice,rats or rabbits), more particularly pa, homologues of ATR may beobtained and such homologues and fragments thereof in general will becapable of selectively hybridizing to Seq. ID No. 1. Such sequences maybe obtained by probing cDNA libraries made from dividing cells ortissues or genomic DNA libraries from other animal species, and probingsuch libraries with probes comprising all or part of Seq. ID. 1 underconditions of medium to high stringency (for example 0.03M sodiumchloride and 0.03M sodium citrate at from about 50° C. to about 60° C.).

[0028] Allelic variants and species homologues may also be obtainedusing degenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences. Conserved sequences can be predicted from aligning the AIRamino acid sequence with that of rad3. The primers will contain one ormore degenerate positions and will be used at stringency conditionslower than those used for cloning sequences with single sequence primersagainst known sequences.

[0029] Alternatively, such polynucleotides may be obtained by sitedirected mutagenesis of the ATR sequences or allelic variants thereof.This may be useful where for example silent codon changes are requiredto sequences to optimise codon preferences for a particular host cell inwhich the polynucleotide sequences are being expressed. Other sequencechanges may be desired in order to introduce restriction enzymerecognition sites, or to alter the property or function of thepolypeptides encoded by the polynucleotides. Further changes may bedesirable to represent particular coding changes found in ATR which giverise to mutant AIR genes which have lost the checkpoint fiction. Probesbased on such changes can be used as diagnostic probes to detect suchAIR mutants.

[0030] The invention further provides double stranded polynucleotidescomprising a polynucleotide of the invention and its complement.

[0031] Polynucleotides or primers of the invention may carry a revealinglabel. Suitable labels include radioisotopes such as ³²P or ³⁵S, enzymelabels, or other protein labels such as biotin. Such labels may be addedto polynucleotides or primers of the invention and may be detected usingby techniques known per se.

[0032] Polynucleotides or primers of the invention or fragments thereoflabelled or unlabelled may be used by a person skilled in the art innucleic acid-based tests for detecting or sequencing ATR in the human oranimal body.

[0033] Such tests for detecting generally comprise bringing a human oranimal body sample contain DNA or RNA into contact with a probecomprising a polynucleotide or primer of the invention under hybridizingconditions and detecting any duplex formed between the probe and nucleicacid in the sample. Such detection may be achieved using techniques suchas PCR or by immobilizing the probe on a solid support, removing nucleicacid in the sample which is not hybridized to the probe, and thendetecting nucleic acid which has hybridized to the probe. Alternatively,the sample nucleic acid may be immobilized on a solid support, and theamount of probe bound to such a support can be detected. Suitable assaymethods of this any other formats can be found in for example WO89/03891and WO90/13667.

[0034] Tests for sequencing ATR include bringing a human or animal bodysample containing target DNA or RNA into contact with a probe comprisinga polynucleotide or primer of the invention under hybridizing conditionsand determining the she by, for example the Sanger dideoxy chaintermination method (see Sambrook et al.).

[0035] Such a method generally comprises elongating, in the presence ofsuitable reagents, the primer by synthesis of a stand complementary tothe target DNA or RNA and selectively terminating the elongationreaction at one or more of an A, C, G or T/U residue; allowing standelongation and termination reaction to occur; separating out accordingto size the elongated products to determine the sequence of thenucleotides at which selective tenon has occurred. Suitable reagentsinclude a DNA polymerase enzyme, the deoxynucleotides dATP, dCTP, dGTPand dTTP, a buffer and ATP. Dideoxynucleotides are used for selectivetermination.

[0036] Tests for detecting or sequencing ATR in the human or animal bodymay be used to determine ATR sequences within cells in individuals whohave, or are suspected to have, an altered ATR gene sequence, forexample within cancer cells including leukaemic cells and solid tumourssuch as breast, ovary, lung, colon, pancreas, testes, liver, brain,muscle and bone tumours.

[0037] In addition, the discovery of ATR will allow the role of thisgene in hereditary diseases to be investigated, in a manner analogous tothe ATM gene. In general, this will involve establishing the status ofATR (e.g using PCR sequence analysis) in cells derived from patientswith diseases that may be connected with damage to replicating cells,e.g. familial predisposition to cancer. chromosome breakage orinstability phenotype or repair-damage sensitivity phenotype.

[0038] The probes of the invention may conveniently be packaged in theform of a test kit in a suitable container. In such kits the probe maybe bound to a solid support where the assay format for which the kit isdesigned requires such binding. The kit may also contain suitablereagents for treating the sample to be probed, hybridizing the probe tonucleic acid in the sample, control reagents, instructions, and thelike.

[0039] The present invention also provides polynucleotides encoding thepolypeptides of the invention described below. Because suchpolynucleotides will be useful as sequences for recombinant productionof polypeptides of the invention, it is not necessary for them to beselectively hybridizable to the sequence Seq. ID No. 1, although thiswill generally be desirable. Otherwise, such polynucleotides may belabelled, used, and made as described above if desired. Polypeptides ofthe invention are described below.

[0040] Particularly preferred polynucleotides of the invention are hosederived from the lipid kinase domain of AIR, its allelic variants andspecies homologues. The lipid kinase domain is represented bynucleotides 7054 to 8011 of Seq. ID. 1. Polynucleotides of the inventionwhich comprise this domain are particularly preferred. The “lipid kinasedomain” refers to a domain which has homology to other known lipidkinases, in particular the p110 subunit of PI-3 kinase, as determined bysequence alignments.

[0041] Other preferred polynucleotides of the invention those whichcomprise nucleotides encoding amino acids 181 to 302 of Seq. ID No. 2(nucleotides 620 to 985 of Seq. ID No. 1), which is believed to be aleucine zipper region, a putative site of protein-protein interaction,and amino acids 1358 to 1366 (nucleotides 4151 to 4177), which is alsoconserved. In an additional aspect, polynucleotides of the inventioninclude those of Seq. ID No. 3 and fragments thereof capable ofselectively hybridizing to this sequence other than the fragmentconsisting of nucleotides 2482 to 6599 in which the following changeshave been made: Deletion of residues 2499, 2501, 2507 & 2509; insertionof C between 5918/5919.

[0042] Particularly preferred fragments include those comprisingresidues 6826 to 7334 (the lipid kinase domain) and the leucine zipperregions 1476 to 1625 and 2310 to 2357. Additionally, the fragmentcomprising the conserved region 3891 to 3917 is preferred. Suchpolypeptides and fragments may be made and used as described above.

[0043] B. Polypeptides.

[0044] Polypeptides of the invention include polypeptides insubstantially isolated form which comprise the sequence set out in SeqID No. 2.

[0045] Polypeptides further include van of such sequences, includingnaturally occurring allelic variants and synthetic variants which aresubstantially homologous to said polypeptides. In this context,substantial homology is regarded as a sequence which has at least 70%,e.g. 80% or 90% amino acid homology (identity) over 30 amino acids withthe sequence of Seq. ID No. 2 except for the lipid kinase domain andC-terminal portion (residues 2326 to 2644) where substantial homology isregarded as at least 80% homology, preferably 90% homology (identity)over 50 amino acids.

[0046] Polypeptides also include other those encoding AIR homologuesfrom other species including animals such as mammals (e.g. mice, rats orrabbits), especially primates, and variants thereof as defined above.

[0047] Polypeptides of the invention also include fragments of the abovementioned full length polypeptides and variants thereof, includingfragments of the sequence set out in Seq. ID No. 2.

[0048] Preferred fragments include those which include an epitope,especially an epitope. Suitable fragments will be at least about 5, e.g.10, 12, 15 or 20 amino acids in size. Polypeptide fragments of the ATRprotein and allelic and species variants thereof may contain one or more(e.g. 2, 3, 5, or 10) substitutions, deletions or insertions, includingconserved substitutions.

[0049] Conserved substitutions may be made according to the followingtable indicates conservative substitutions, where amino acids on thesame block in the second column and preferably in the same line in thethird column may be substituted for each other: ALIPHATIC Non-polar G AP I L V Polar - uncharged C S T M N Q Polar - charged D E K R AROMATIC HF W Y OTHER N Q D E

[0050] Variants of the polypeptides of the invention may also comprisepolypeptides wherein one or more of the specified (i.e., naturallyencoded) amino acids is deleted or replaced or wherein one or morenonspecified ammo acids arc added: (1) without loss of the kinaseactivity specific to the polypeptides of the invention; or (2) withdisablement of the kinase activity specific to the polypeptides of theinvention; or (3) with disablement of the ability to intend with membersor regulators of the cell cycle checkpoint pathway.

[0051] Epitopes may be determined either by techniques such as peptidescanning techniques as described by Geysen et al. Mol. Immunol., 2;709-715 (1986).

[0052] Polypeptides of the invention may be in a substantially isolatedform. It will be understood that the polypeptide may be mixed withcarriers or diluents which will not interfere with the intended purposeof the polypeptide and still be regarded as substantially isolated. Apolypeptide of the invention may also be in a substantially purifiedform, in which case it will generally comprise the polypeptide in apreparation in which more than 90%, e.g. 95%, 98% or 99% of thepolypeptide in the preparation is a polypeptide of the invention.Polypeptides of the invention may be modified for example by theaddition of Histidine residues to assist their purification or by theaddition of a signal sequence to promote their secretion from a cell.

[0053] A polypeptide of the invention may be labelled with a revealinglabel. The revealing label may be any suitable label which allows thepolypeptide to be detected. Suitable labels include radioisotopes, e.g.¹²⁵I, enzymes, antibodies, polynucleotides and linkers such as biotin.Labelled polypeptides of the invention may be used in diagnosticprocedures such as immunoassays in order to determine the amount of apolypeptide of the invention in a sample. Polypeptides or labelledpolypeptides of the invention may also be used in serological or cellmediated immune assays for the detection of immune reactivity to saidpolypeptides in animals and humans using standard protocols.

[0054] A polypeptide or labelled polypeptide of the invention orfragment thereof may also be fixed to a solid phase, for example thesurface of an immunoassay well or dipstick.

[0055] Such labelled and/or immobilized polypeptides may be paged intokits in a suitable container along with suitable reagents, controls,instructions and the like.

[0056] Such polypeptides and kits may be used in methods of detection ofantibodies to the ATR protein or its allelic or species variants byimmunoassay.

[0057] Immunoassay methods are well known in the an and will generallycomprise:

[0058] (a) providing a polypeptide comprising an epitope bindable by anantibody against said protein;

[0059] (b) incubating a biological sample with said polypeptide underconditions which allow for the formation of an antibody-antigen complex;and

[0060] (c) determining whether antibody-antigen complex comprising saidpolypeptide is formed.

[0061] Polypeptides of the invention may be may by synthetic means (e.g.as described by Geysen et al.) or recombinantly, as described below.

[0062] Particularly preferred polypeptides of the invention includethose spanning or within the lipid kinase domain, namely from aminoacids 2326 to 2644 of Seq. ID. 2. or sequences substantially homologousthereto. Fragments as defined above from this region are particularlypreferred. The polypeptides and fragments thereof may contain amino acidalterations as defined above, including substitutions at one or more ofpositions 2475, 2480 and 2494, which correspond to the positions of therad3 substitutions described in the examples below. Preferredsubstiutions include D2475A, N2480K and D2494E.

[0063] Polypeptides of the invention may be used in in vitro or in vivocell culture systems to study the role of AIR as a checkpoint gene. Forexample, truncated or modified (e.g. modified in the lipid kinasedomain) ATRs may be introduced into a cell to disrupt the normalcheckpoint functions which occur in the cell.

[0064] The polypeptides of the invention may be introduced into the cellby in situ expression of the polypeptide from a recombinant expressionvector (see below). The expression vector optionally carries aninducible promoter to control the expression of the polypeptide.

[0065] The use of mammalian host cells is expected to provide for suchpost-translational modifications (e.g., myristolation, glycosylation,truncation, lapidation and tyrosine, serine or threoninephosphorylation) as may be needed to confer optimal biological activityon recombinant expression products of the invention.

[0066] Such cell culture systems in which polypeptide of the inventionare expressed may be used in assay systems to identify candidatesubstances which interfere or enhance checkpoint functions in the cell(see below).

[0067] In an additional aspect, polypeptides of the invention includethe protein of Seq. ID No. 4 and fragments thereof from the region otherthan the fragment consisting of amino acids 713 to 1778. Particularlypreferred fragments include those comprising residues 2082 to 2386 (thelipid kinase domain) and the leucine zipper regions 298 to 347 and 576to 591. Additionally, the fragment comprising the conserved region 1103to 1111 is preferred. Such polypeptides and fragments may be made andused as described above.

[0068] The invention also provides polypeptides substantially homologousto the protein of Seq. ID No. 4, and fragments thereof. In this context,substantial homology is regarded as a sequence which has at least 70%,e.g. 80% or 90% amino acid homology (identity) over 30 amino acids withthe sequence of Seq. ID No. 4 except for the lipid kinase domain andC-terminal portion (residues 2082 to 2386) where substantial homology isregarded as at least 80%, preferably at least 90% homology (identity)over 50 amino acids.

[0069] C. Vectors.

[0070] Polynucleotides of the invention can be incorporated into arecombinant replicable vector. The vector may be used to replicate thenucleic acid in a compatible host cell. Thus in a further embodiment,the invention provides a method of making polynucleotide of theinvention by introducing a polynucleotide of the invention into areplicable vector, introducing the vector into a compatible host cell,and growing the host cell under conditions which bring about replicationof the vector. The vector may be recovered from the host cell. Suitablehost cells are described below in connection with expression vectors.

[0071] D. Expression Vectors.

[0072] Preferably, a polynucleotide of the invention in a vector isoperably linked to a control sequence which is capable of providing forthe expression of the coding sequence by the host cell, i.e. the vectoris an expression vector.

[0073] The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

[0074] Such vectors may be transformed into a suitable host cell asdescribed above to provide for expression of a polypeptide of theinvention. Thus, in a further aspect the invention provides a processfor preparing polypeptides according to the invention which comprisescultivating a host cell transformed or transfected with an expressionvector as described above under conditions to provide for expression bythe vector of a coding sequence encoding the polypeptides, andrecovering the expressed polypeptides.

[0075] The vectors may be for example, plasmid, virus or phage vectorsprovided with an origin of replication, optionally a promoter for theexpression of the said polynucleotide and optionally a regulator of thepromoter. The vectors may contain one or more selectable marker genes,for example an ampicillin resistance gene in the case of a bacterialplasmid or a neomycin resistance gene for a mammalian vector. Vectorsmay be used in vitro, for example for the production of RNA or used totransfect or transform a host cell. The vector may also be adapted to beused in vivo, for example in a method of gene therapy.

[0076] A further embodiment of the invention provides host cellstransformed or transfected with the vectors for the replication andexpression of polynucleotides of the invention. The cells will be chosento be compatible with the said vector and may for example be bacterial,yeast, insect or mammalian.

[0077] Polynucleotides according to the invention may also be insertedinto the vectors described above in an antisense orientation in order toprovide for the production of antisense RNA. Antisense RNA or otherantisense polynucleotides may also be produced by synthetic means. Suchantisense polynucleotides may be used in a method of controlling thelevels of ATR or its variants or species homologues.

[0078] Promoters and other expression regulation signals may be selectedto be compatible with the host cell for which the expression vector isdesigned. For example, yeast promoters include S. cerevisiae GAL4 andADH promoters, S. pombe nmt1 and adh promoter. Mammalian promotersinclude the metallothionein promoter which is can be included inresponse to heavy metals such as cadmium. Viral promoters such as theSV40 large T antigen promoter or adenovirus promoters may also be used.All these promoters are readily available in the art.

[0079] E. Antibodies.

[0080] The invention also provides monoclonal or polyclonal antibodiesto polypeptides of the invention or fragments thereof. The inventionfurther provides a process for the production of monoclonal orpolyclonal antibodies to polypeptides of the invention. Monoclonalantibodies may be prepared by conventional hybridoma technology usingthe polypeptides of the invention or peptide fragments thereof, asimmunogens. Polyclonal antibodies may also be prepared by conventionalmeans which comprise inoculating a host animal, for example a rat or arabbit, with a polypeptide of the invention or peptide fragment thereofand recovering immune serum.

[0081] In order that such antibodies may be made, the invention alsoprovides polypeptides of the invention or fragments thereof haptenisedto another polypeptide for use as immunogens in animals or humans

[0082] Preferred antibodies of the invention will be capable ofselectively binding the human ATR protein, that is with an affinity atleast 10 fold, preferably at least 100 fold that of the rad3 protein.Such antibodies can be obtained by routine experimentation, e.g.selecting regions of ATR protein with sequences different from thecorresponding regions of rad, making peptides comprising such sequencesand using such peptides as immunogens. Following production ofantibodies the binding of said antibodies may be determined. Preferredantibodies of the invention include those capable of selectively bindingthe lipid kinase domain (as defined above) of the human ATR protein. Inaddition, antibodies which are capable of binding the human and yeast(S. pombe) lipid kinase domains with similar affinity, but not to thedomains of the ATM family of proteins form a further aspect of theinvention. Such antibodies may be raised against peptides from the lipidse domains which correspond to regions found to be identical, orsubstantially identical, in the yeast and human genes.

[0083] For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes fragments of whole antibodies whichretain their binding activity for a tumour target antigen. Suchfragments include Fv, F(ab′) and F(ab′)₂ fragments, as well as singlechain antibodies. Furthermore, the antibodies and fragments thereof maybe humanised antibodies, eg. as described in EP-A-239400.

[0084] Antibodies may be used in method of detecting polypeptides of theinvention present in biological samples by a method which comprises:

[0085] (a) providing an antibody of the invention;

[0086] (b) incubating a biological sample with said antibody underconditions which allow for the formation of an antibody-antigen complex;and

[0087] (c) determining whether antibody-antigen complex comprising saidantibody is formed.

[0088] Suitable samples include extracts from dividing cells, e.gleukocytes or cancer cells including leukaemic cells and solid tumourssuch as breast, ovary, lung, colon, pancreas, testes, liver, brain,muscle and bone tumours.

[0089] Antibodies of the invention may be bound to a solid supportand/or packaged into kits in a suitable container along with suitablereagents, controls, instructions and die like.

[0090] F. Assays.

[0091] Abrogating cell cycle checkpoints is a potential strategy fordeveloping or designing drugs for anti cancer therapy, both as a noveltreatment as such and as part of a combination therapy to enhance thespecific toxicity of current chemotherapeutic agents. For examplealkylating agents such as nitrogen mustards are used a chemotherapeuticagents which damage DNA in rapidly dividing cells, leading to celldeath. The toxicity of such agents may be lessened by DNA repair andcheckpoint mechanisms. Abrogating such mechanisms will thus enhance theeffectiveness of therapeutic compounds designed to damage DNA.Abrogation of the ATR checkpoint will be especially useful where tumourcells have lost other checkpoint or damage response genes, since theseother genes may be able to complement the loss of ATR function in nontumour cells, leading to an even greater enhancement in theeffectiveness of the chemotherapeutic agent.

[0092] The lipid kinase activity of ATR is a target for developinganticancer compounds, since the results presented in the followingexamples indicate that the kinase domain is required for ATR function.Thus the present invention provides an assay method for screeningcandidate substances for anti-cancer therapy which comprises:

[0093] (a) providing a polypeptide of the invention which retains lipidkinase activity and a substrate for said kinase, under conditions andwith reagents such that the kinase activity will act upon the substrate;

[0094] (b) bringing said polypeptide and substrate into contact with acandidate substance;

[0095] (c) measuring the degree of decrease in the kinase activity ofthe polypeptide; and

[0096] (d) selecting a candidate substance which provides a decrease inactivity.

[0097] The assay may be carried out in vitro, for example in the wellsof a microtitre dish. Such a format may be readily adapted forautomation, allowing large numbers of candidate substances to bescreened.

[0098] The substrate may be a protein or lipid substrate of natural orsynthetic origin upon which the polypeptide of the invention will act.Usually, the polypeptide of the invention will phosphorylate thesubstrate.

[0099] Any suitable format for the assay may be used by those of skillin the art of throughput assays. Typically, the polypeptide of theinvention which retains lipid kinase activity will be bound to a solidsupport in the presence of a substrate and cellular and other componentswhich are usually required for activity. Labelled phosphate and acandidate substance will be added to the mixture simultaneously orsequentially in either order. After a suitable reaction time (usually afew minutes but in any event enough for phosphorylation of the substratein the absence of candidate substance to occur) the amount of freephosphate is determined, e.g. by precipitation of phosphate. Candidatesubstances which inhibit kinase activity will inhibit the incorporationof free phosphate into the substrate and thus where free phosphate isfound this is indicative of inhibition.

[0100] Other assay formats may be used by those skilled in the art.

[0101] The candidate substances may be used in an initial screen inbatches of for example 10 compounds per reaction, and the compounds ofthose batches which show inhibition tested individually.

[0102] Suitable candidate substances include peptides, especially offrom about 5 to 20 amino acids in size, based on the sequence of thekinase domain, or variants of such peptides in which one or moreresidues have been substituted as described above. Peptides from panelsof peptides comprising random sequences or sequences which have beenvaried consistently to provide a maximally diverse panel of peptides maybe used. Further candidate substances include kinase inhibitors whichare small molecules such as cyclosporin-like and staurosporin-likecompounds, or other compounds commercially available in panels of smallmolecule inhibitors.

[0103] Candidate substances which show activity in in vitro screens suchas the above can then be tested in in vivo systems, such as yeast ormammalian cells which will be exposed to the inhibitor and tested forcheckpoint activity.

[0104] We have also shown that Rad3 possesses protein kinase activity.Target substrates of Rad3 protein kinase activity may be identified byincorporating test compounds in assays for kinase activity. Rad3 proteinis resuspended in kinase buffer and incubated either in the presence ofabsence of the test compound (e.g., casein, histone H1, or appropriatesubstrate peptide). Moles of phosphate transferred by the kinase to thetest compound are measured by autoradiography or scintillation courting.Transfer of phosphate to the test compound is indicative that the testcompound is a substrate of the kinase.

[0105] Agents that modulate Rad3/ATR lipid kinase or Rad 3 proteinkinase activity may be identified by incubating a test compound andRad3/ATR immunopurified from cells naturally expressing Rad3/ATR, withRad3/ATR obtained from recombinant procaryotic or eukaryotic cellsexpressing the enzyme, or with purified Rad3/ATR, and then determiningthe effect of the test compound on Rad3/ATR activity. The activity ofthe Rad3/ATR lipid kinase or Rad3 protein kinase domains can be measuredby determining the moles of ³²P-phosphate transferred by the kinase fromgamma-³²-P-ATP to either itself (autophosphorylation) or to an exogenoussubstrate such as a lipid or protein. The amount of phosphateincorporated into the substrate is measured by scintillation counting orautoradiography. An increase in the moles of phosphate transferred tothe substrate in the presence of the test compound compared to the molesof phosphate transferred to the substrate in the absence of the testcompound indicates that the test compound is an activator of said kinaseactivity. Conversely, a decrease in the moles of phosphate transferredto the substrate in presence of the test compound compared to the molesof phosphate transferred to the substrate in the absence of the testcompound indicates that the modulator is an inhibitor of said kinaseactivity.

[0106] In a presently preferred assay, a Rad3/ATR antibody linked toagarose beads is incubated with a cell lysate prepared from host cellsexpressing Rad3/ATR. The beads are washed to remove proteins bindingnonspecifically to the beads and the beads are then resuspended in akinase buffer (such as 25 mM K-HEPES pH 7.7, 50 mM potassium chloride,10 mM magnesium chloride. 0.1% Nonidet-P40, 20 % glycerol, 1 mM DTT).The reaction is initiated by the addition of 100 μM gamma-³²P-ATP (4Ci/mM) and an exogenous substrate such as lipid or peptide, and thereaction is carried out at 30° C. for 10 minutes. The activity of thekinase is measured by determining the moles of ³²P-phosphate transferredeither to the kinase itself or the added substrate. In a preferredembodiment the host cells lack endogenous Rad3/ATR kinase activity. Theselectivity of a compound that modulates the lipid kinase activity ofRad3/ATR can be evaluated by comparing its activity on Rad3/ATR to itsactivity on, for example, other known phosphatidylinositol-3(PI-3)related kinases. The combination of the recombinant Rad3/ATRproducts of the invention with other recombinant PI-3-related kinaseproducts in a series of independent assays provides a system fordeveloping selective modulators of Rad3/ATR kinase activity. Similarly,the selectivity of a compound that modulates the protein kinase activityof Rad3 may be determined with reference to other protein kinases, forexample the DNA dependent protein kinase or ATM.

[0107] In addition, the demonstration that the rad mutant rad.D2249E(see Examples) can act as a dominant negative mutant indicatesinvolvement in one or more protein complexes, and such complexesthemselves can be targeted for therapeutic interventions We have shown,for example, that Rad3 can both self associate and associate with ATR.It is therefore likely that Rad/ATR function as multimeric moleculesMutant yeast rad or human ATR genes, or derivatives thereof which alsolack rad/ATR activity may be introduced into cells to act as dominantnegative mutants. Thus for example if expression of a dominant negativemutant (e.g. ATR D2475A, N2480K or D2494E) in a tumour cell leads toenhanced radiation sensitivity this indicates that the native ATR isstill functioning and thus a target for therapeutic agents.

[0108] Interacting proteins including components of multimeric proteincomplexes involving Rad3 or ATR may be identified by the followingassays.

[0109] A first assay contemplated by the invention is a two-hybridscreen. The two-hybrid system was developed in yeast (Chien et al.(1991)) and is based on functional in vivo reconstitution of atranscription factor which activates a reporter gene. Specifically, apolynucleotide encoding a protein that interacts with Rad3/ATR isisolated by: transforming or transfecting appropriate host cells with aDNA construct comprising a reporter gene under the control of a promoterregulated by a transcription factor having DNA a binding domain and anactivating domain: expressing in the host cells a first hybrid DNAsequence encoding a fire fusion of part or all of Rad3/ATR and eitherthe DNA binding domain or the activating domain of the transcriptionfactor; expressing in the host cell a library of second hybrid DNAsequences encoding second fusion of part or all putative Rad3/ATRbinding proteins and the DNA binding domain or activating domain of thetranscription factor which is not incorporated in the first fusion;detecting binding of an Rad3/ATR interacting protein to Rad3/ATR in aparticular host cell by detecting the production of reporter geneproduct in the host cell; and isolating second hybrid DNA sequencesencoding the interacting protein from the particular host cell.Presently preferred for use in the assay are a lexA promoter to driveexpression of the reporter gene, the reporter gene, a transcriptionfactor comprising the lexA DNA binding domain and the GAL4transactivation domain, and yeast host cells.

[0110] Other assays for identifying proteins that interact with Rad3 orATR may involve immobilising Rad3/ATR or a test protein, detectablylabelling the nonimmobilised binding partner, incubating the bindingpartners together and determining the amount of label bound. Bound labelindicates that the test protein interacts with Rad3/ATR.

[0111] Another type of assay for identifying Rad3 or ATR interactingproteins involves immobilising Rad3/ATR or a fragment thereof on a solidsupport coated (or impregnated with) a fluorescent agent, labelling atest protein with a compound capable of exciting the fluorescent agent,contacting the immobilised Rad3/ATR with the labelled test protein,detecting light emission by the fluorescent agent, and identifyinginteracting proteins as test proteins which result in the emission oflight by the fluorescent agent. Alternatively, the putative interactingprotein may be immobilised and Rad3/ATR may be labelled in the assay.

[0112] Compounds that modulate interaction between Rad3/ATR and othercellular components may be used in methods of treating cancer. Forexample, if a particular form of cancer results from a mutation in agene other than ATR such as the p53 gene, an agent which inhibits thetranscription or the enzymatic activity of AIR and thus the G₂ cellcycle checkpoint may be used to render cancerous cells more susceptibleto chemotherapy or radiation therapy. The therapeutic value of such anagent lies in the fact that current radiation therapy or chemotherapy inmost cases does nothing to overcome the ability of the p53 mutantcancerous cell to sense and correct the DNA damage imposed as a resultof the treatment. As a result, a cancer cell can simply repair the DNAdamage. Modulating agents of the invention may therefore be chemotherapyand radiation adjuvants or may be directly active as chemotherapy drugsthemselves.

[0113] Assays for identifying compounds that modulate interaction ofRad3/ATR with other proteins may involve: transforming or transfectingappropriate host cells with a DNA construct comprising a reporter geneunder the control of a promoter regulated by a transcription factorhaving a DNA-binding domain and an activating domain: expressing in thehost cells a first hybrid DNA sequence encoding a first fusion of partor all of Rad3/ATR and the DNA binding domain or the activating domainof the transcription factor; expressing in the host cells a secondhybrid DNA sequence encoding part or all of a protein that interactswith Rad3/ATR and the DNA binding domain or activating domain of thetranscription factor which is not incorporated in the first fusion;evaluating the effect of a test compound on the interaction betweenRad3/ATR and the interacting protein by detecting binding of theinteracting protein to Rad3/ATR in a particular host cell by measuringthe production of reporter gene product in the host cell in the presenceor absence of the test compound; and identifying modulating compounds asthose test compounds altering production of the reported gene product incomparison to production of the reporter gene product in the absence ofthe modulating compound. Presently preferred for use in the assay are alexA promoter to drive expression of the reporter gene, the lacZreporter gene, a transcription factor comprising the lexA DNA domain andthe GALA transactivation domain, and yeast host cells.

[0114] Another type of assay for identifying compounds that modulate theinteraction between Rad3/ATR and an interacting protein involvesimmobilising Rad3/ATR or a natural Rad3/ATR interacting protein,detectably labelling the nonimmobilised binding partner, incubating thebinding partners together and determining the effect of a test compoundon the amount of label bound wherein a reduction in the label bound inthe present of the test compound compared to the amount of label boundin the absence of the test compound indicates that the test agent is aninhibitor of Rad3/ATR interaction with the protein. Conversely, anincrease in the bound in the presence of the test compared to the amountlabel bound in the absence of the compared indicates that the putativemodulator is an activator of Rad3/ATR interaction with the protein.

[0115] Yet another method contemplated by the invention for identifyingcompounds that modulate the binding between Rad3/ATR and an interactingprotein involves immobilising Rad3/ATR or a fragment thereof on a solidsupport coated (or impregnated with) a fluorescent agent, labelling theinteracting protein with a compound capable of exciting the fluorescentagent, contacting the immobilised Rad3/ATR with the labelled interactingprotein in the presence and absence of a test compound, detecting lightemission by the fluorescent agent, and identifying modulating compoundsas those test compounds that affect the emission of light by thefluorescent agent in comparison to the emission of light by thefluorescent agent in the absence of the test compound. Alternatively,the Rad3/ATR interacting protein may be immobilised and Rad3/ATR may belabelled in the assay.

[0116] We have shown that Rad3 interacts with ATR. Therefore theabove-mentioned assays may also be used to identify compounds thatmodulate the interaction between Rad3 and AT where the interactingprotein described in the assay methods is either Rad3 or ATR.

[0117] We have also shown that Rad3 can bind to itself, stronglysuggesting that ATR can also bind to itself. Therefore theabove-mentioned assays may also be used to identify compounds thatmodulate Rad3-Rad3 interactions and AIR-ATR interactions.

[0118] Such compounds could be used therapeutically to disrupt ATR-ATRinteractions and increase the sensitivity of tumour cells tochemotherapy and/or radiotherapy. Thus the invention provides an assaymethod for screening candidate substances for anti cancer therapy whichcomprises:

[0119] (a)

[0120] (i) incubating a polypeptide of the invention with anotherpolypeptide of the invention, which may be the same as or different tothe first polypeptide, under conditions which allow the firstpolypeptide to bind to the second polypeptide to form a complex;

[0121] (ii) bringing the complex thus formed into contact with acandidate substance; or

[0122] (a) incubating a polypeptide of the invention with anotherpolypeptide of the invention, which may be the same as or different tothe first polypeptide, under conditions which allow the firstpolypeptide to bind to the second polypeptide to form a complex and inthe presence of a candidate substance; and

[0123] (b) determining whether the candidate substance inhibits bindingof the first polypeptide to the second polypeptide and

[0124] (c) selecting a candidate substance which inhibits binding of thefirst polypeptide to the second polypeptide.

[0125] Preferably the first and second polypeptide may be distinguishedfrom each other. For example, the first polypeptide and the secondpolypeptide may both be ATR, or may both be Rad3, or one may be ATR andone may be Rad3 or derivatives of either ATR or Rad3 which retainbinding activity. When both polypeptides are AIR or Rad3. preferably twodistinguishable forms of ATR/Rad3 would be used in these assays. Theymay be distinguished by, for example, labelling either of thepolypeptides. Examples of labels include radioactive labels. epitopetags or other polypeptide tags such as glutathione-S-transferase. Forexample, one form of Rad3 may have one form of epitope tag, and theother form would have a different epitope tag, allowing them to bedistinguished immunologically such that binding of one to the other canbe ascertained quantitively or qualitatively. In a preferred method, thefirst polypeptide may be immobilised, for example to agarose beads or asolid support, and the second polypeptide may be in free solution.Binding is then determined using methods described above and well-knownto skilled persons.

[0126] Also comprehended by the present invention are antibody products(e.g., monoclonal and polyclonal antibodies, single chain antibodies,chimeric antibodies, CDR-grafted antibodies and the like) and otherbinding proteins (such as those identified in the assays above) whichare specific for the Rad3 protein kinase domain or the Rad3/ATR lipidkinase domains. Binding proteins can be developed using isolated naturalor recombinant enzymes. The binding proteins are useful, in turn, forpurifying recombinant and naturally occurring enzymes and identifyingcells producing such enzymes. Assays for the detection andquantification of proteins in cells and in fluids may involve a singleantibody substance or multiple antibody substances in a “sandwich” assayformat. The binding proteins are also manifestly useful in modulating(i.e., blocking, inhibiting, or stimulating) enzyme/substrate orenzyme/regulator interactions.

[0127] Modulators of Rad3/ATR may affect its kinase activity, itslocalisation in the cell, and/or its interaction with members of thecell cycle checkpoint pathway. Selective modulators may include, forexample, polypeptides or peptides which specifically bind to Rad3/ATR orRad3/ATR nucleic acid, and/or other non-peptide compounds (e.g.,isolated or synthetic organic molecules) which specifically react withRad3/ATR or Rad3/ATR nucleic acid. Mutant forms of Rad3/ATR which affectthe enzymatic activity or cellular localisation of wild-type Rad3/ATRare also contemplated by the invention.

[0128] Furthermore, combinatorial libraries, peptide and peptidemimetics, defined chemical entities, oligonucleotides, and naturalproduct libraries may be screened for activity as modulators of Rad3/ATRkinase activity and RAd3/ATR interactions in assays such as thosedescribed

[0129] F. Therapeutic uses

[0130] Modulators of RAd3/ATR activity, including inhibitors of theirlipid kinase and protein kinase activities, may be used in anti-cancertherapy. In particular, they may be used to increase the susceptibilityof cancer cells to chemotherapy and/or radiotherapy by virtue of theirability to disrupt the cell cycle regulatory functions of Rad3/ATR.

[0131] Thus the invention provides the use of compounds that modulateRad3/ATR activity, identified by the screening assays described above,in a method of treatment of cancer. In one embodiment, said compoundsare capable of inhibiting rad3/ATR lipid kinase and/or Rad3 proteinkinase activity. In another embodiment, said compounds are capable ofinhibiting interactions between ATR and itself and/or between ATR andother interacting proteins which may, for example, normally form part ofa multimeric protein complex.

[0132] It is to be understood that the term “compound” in this contextalso refers 10 the candidate substances selected in the above-describedassays.

[0133] Typically the compounds are formulated for clinicaladministration by mixing them with a pharmaceutically acceptable carrieror diluent. For example they can be formulated for topical, parenteral,intravenous, intramuscular, subcutaneous, intraocular or transdermaladministration. Preferably, the compound is used in an injectable form.Direct injection into the patient's tumour is advantageous because itmakes it possible to concentrate the therapeutic effect at the level ofthe affected tissues. It may therefore be so with any vehicle which ispharmaceutically acceptable for an injectable formulation, preferablyfor a direct injection at the site to be treated. The pharmaceuticallycarrier or diluent may be, for example, sterile or isotonic solutions.

[0134] The dose of compound used may be adjusted according to variousparameters, especially according to the compound used, the age, weightand condition of the patient to be treated, the mode of administrationused, pathology of the tumour and the required clinical regimen. As aguide, the amount of compound administered by injection is suitably from0.01 mg/kg to 30 mg/kg, preferably from 0.1 mg/kg to 10 mg/kg.

[0135] The routes of administration and dosages described are intendedonly as a guide since a skilled practitioner will be able to determinereadily the optimum route of administration and dosage for anyparticular patient and condition.

[0136] Compounds to be administered may include polypeptides or nucleicacids. The nucleic acids may encode polypeptides or they may encodeantisense constructs that inhibit expression of a cellular gene. Nucleicacids may be administered by, for example, lipofection or by viralvectors. For example, the nucleic acid may form part of a viral vectorsuch as an adenovirus. When viral vectors are used, in general the doseadministered is between 10⁴ and 10¹⁴ pfu/ml, preferably 10⁶ to 10¹⁰pfu/ml. The term pfu (“plaque forming unit”) corresponds to theinfectivity of a virus solution and is determined by infecting anappropriate cell culture and measuring, generally after 48 hours, thenumber of plaques of infected cells. The techniques for determining thepfu titre of a viral solution are well documented in the literature.

[0137] Any cancer types may be treated by these methods, for exampleleukaemias, and solid tumours such as breat, ovary, lung, colon,pancreas, testes, liver, brain, muscle and bone tumour. Preferably, thetumour has normal ATR function.

DESCRIPTION OF THE DRAWINGS

[0138]FIG. 1

[0139] The relationship between ATR. rad3, mei-41, MEC1, TEL1 and ATM

[0140] A. Overall structures of ATR, Rad3, Mei-41, Mec1p, Te11p and ATM.

[0141] Legend: open square—Rad3 domain; hatched boxes—kinase domain

[0142] B. Dendrogram based on sequence alignments generated by theClustal method (PAM250) using DNAstar software, rad3ESR1/mei-41/ATR aremore closely related to each other than to ATM and TEL1. Sequences ofrad3 and ATM are available in the EMBL database.

[0143] The following examples illustrate the invention.

EXAMPLE 1

[0144] The rad3 gene of S. pombe is one of six genes absolutely requiredfor the DNA structure checkpoints in . pombe (Al-Khodairy and Carr,1992; Al-Khodairy et a. 1994). A sequence refining part of the rad3 genewas reported by Seaton et al. (1992). In attempting to clarify theintron/exon structure of this gene we identified sequencing anomalies atboth the 5′ and 3′ ends. We have sequenced the complete gene (seeExperimental Procedures) and find that rad3 is capable of encoding aproduct of 2386 amino acids. The C-terminal region contains theconsensus sequences typical of a sub-class of kinases known as lipidkinases, the founder member of which is the p110 catalytic subunit ofPI3 kinase (Hiles et al. 1992).

[0145] A truncated rad3 clone lacking the amino terminus and the kinaseregion has been reported to complement the rad3::pR3H1.0 gene disruptionmutant of rad3 (Jimenez et al. 1992). This disruption mutant does notremove the potential kinase domain. To clarify the role of this domain,we have created a null mutant by gene replacement. This mutant has aminoacids 1477-2271 of rad3, including the kinase consensus domain, replacedby ura4⁺. This strain rad3.d, has identical checkpoint defects andradiation/hydroxyurea sensitivities to the rad3.136 mutant (Nasim andSmith. 1975) and the original rad3::pR3H1.0 disruption mutant (Jimenezet at. 1992: Seaton et al. 1992) (data not shown). We have created threeseparate point mutants in the putative kinase domain of rad3 and usedthese in gene replacement experiments to construct strains with definedkinase null mutations. All three stains, rad3.D2230A. rad3.N2235K andrad3.D2249E have phenotypes identical to the rad3.d nu mutant da notshown), suggesting that the kinase activity is required for Rad3function. In the light of our findings, one interpretation of theresults of Seaton et al. (1992) and Jimenez et al. (1992) is that thepartial clone may show intragenic complementation between the plasmidborne truncated gene and a genomic partial deletion which retains kinasefunction. Such an interpretation would be consistent with Rad3 acting asa dimer or multimer.

[0146] When the kinase Dull allele rad3D2249E was moderatelyover-expressed in wild type veils under control of a modified nmt1promoter (Maundrell, 1990), it caused extreme radiation sensitivity,assayed by UV strip tests, and acted as a dominant negative mutant (datanot shown). When the same kinase null construct was expressed at ahigher level, it inhibited growth (data not shown). Examination of thecells indicates that division continued very slowly, and at a smallercell size wild type cells and cells containing empty vector divide atapproximately 15 microns, while rad and rad3.D2249E over-expressingcells divide at approximately 11.2 microns (data not shown). In S.pombe, this usually indicates an advancement of mitosis.

[0147] The human rad3 homolog, ATR

[0148] To identify a human form of rad3, a combination of methods wasapplied. Through these approaches, we have cloned the entire codingregion of a human gene (see materials and methods). which we have namedATR (ataxia and lad related). ATR is capable of encoding a 2644 aminoacid protein which is much more closely related to the products of S.pombe rad3, S. cerevisiae ESR1 (Kato and Ogawa, 1994) and D.melanogaster mei-41 genes (Hari et al. 1995) than to the human ATM andS. cerevisiae Tell proteins (Savitsky et al. 1995; Greenwell et al.1995) and is likely to be the true homolog of rad3. ESR1 is allelic tothe mecI/sad3 checkpoint mutants (Allen et al. 1994; Weinert et al.1994) which have an equivalent phenotype to rad3. ATR is less closelyrelated to the human checkpoint gene ATM, containing C-terminal putativelipid kinase domain and having a similar overall structure. Sequencealignments demonstrate clearly that the rad3/ESR1(MEC1/SAD3)/mei-4/ATRgenes are more closely related to each other than any are to ATR orTEL1, and that ATM is more homologous to TEL1 (FIG. 1).

[0149] The ATM gene is expressed in a wide variety of tissues (Savitskyet al. 1995). In S. cerevisiae, ESR1 shows low level expression inmitotic cells but is rapidly in during meiosis 1 (Kato and Ogawa. 1994).Using Northern blot analysis, we have demonstrated that ATR is alsoweakly expressed in many tissues but that it is mote highly expressed intestis (data not shown). Given that ATP, Rad3 and Esr1p proteins aremore highly related to each other than to ATM, the higher ATR expressionin testis is consistent with the observation that Esr1p has a role inmeiotic recombination (Kato and Ogawa, 1994). Using FISH and PCRanalysis, we have mapped ATR to chromosome 3q22-3q25 (data not shown).This region is not associated with known cancer prone syndromes.

[0150] In order to further investigate the possibility that Rad3 acts asa multimer, we have created two separate tagged constructs of fulllength rad3 in pREP based inducible vectors. In one, Rad3 is translatedwith two myc epitope tags at the N terminus, while in the other theseare substituted for a triple HA epitope tag. When both constructs areexpressed together in wild type cells, it is possible to co-precipitatethe HA tagged Rad3 with the myc specific antibody, and the myc taggedRad3 with the HA specific antibody (data not shown). This demonstratesthat in vivo, the Rad3 protein is capable of self association and isfully consistent with the complementation data of Jimenez et al. (1992).

[0151] Although the ATR gene could not complement the phenotype of therad3 mutants, we have investigated the ability of ATR to form a proteincomplex with S. pombe Rad3 by expressing both ATR and myc-tagged S.pombe Rad3 in the same yeast cells. Using an anti-ATR antibody (whichdoes not precipitate S. pombe Rad3. see materials and methods) we areable to co-precipitate the yeast protein. We were also able toprecipitate the human ATR protein with myc-specific antibodies thatrecognise the S. pombe Rad3 (data not shown). These data suggest thehuman and yeast proteins can form a heteromeric-complex, which supportsthe contention, based on the sequence similarity, of a close functionalrelationship between these homologues.

[0152] Rad3 proteins have associated kinase activity

[0153] Since mutagensis experiments suggest that the kinase activity ofthe Rad3 proteins in vivo appears to be essential for their function, wehave investigated this activity further. Using S. pombe rad3::ura4 cellsexpressing HA tagged S. pombe Rad3, we have been able to detect asignificant protein se activity which precipitates with HA-specificantibodies only when Rad3 is induced and which is not changed followingirradiation (data not shown). This activity, which is specific to Rad3or co-precipitating kinase, appears to reflect phosphorylation of Rad3itself, since the major band above 200 kD that is phosphorylated can bedetected by Western analysis with anti-HA antibody (data not shown).Attempts to identify convenient in vitro substrates such as myelin basicprotein. RP-A and several purified S. pombe checkpoint proteins have sofar proved unsuccessful. When the IP in vitro kinase assay is performedwith cells over-expressing a “kinase-null” D2249E version of Rad3, theassociated kinase activity precipitated by HA-specific antibody issignificantly reduced (data not shown). There are several possibleexplanations for this. The measured kinase activity could reflect Rad3activity directly. In this case the residual activity seen with thekinase dead Rad3 could reflect the fact that it is not unknown for theequivalent D to E mutation in other protein kinases to produce abiologically inert protein with residual in vitro biochemical activity.Alternatively the kinase activity which phosphorylates Rad3 may be dueto associated proteins, and these may interact less effectively with theD2249E mutant protein.

[0154] Discussion

[0155] The checkpoint pathways controlling cell cycle progressionfollowing DNA damage or interference in the individual events whichcomprise the cycle are of considerable importance in maintaining geneticstability and can be considered as pathways which suppress tumorgenesis.Several tumour suppressor genes are intimately involved in subsets ofthe checkpoint pathways (reviewed in Hartwell and Kastan, 1994),particularly those affecting the transition from G1 into S phase andcommitment to the cell cycle. The convergence of the two yeast modelsystems for checkpoints clearly indicates that the genes involved inthese pathways are conserved Our work extends this conservation tometazoan cells, and clarifies the relationship between rad3.ESR1(MEC1/SAD3). mei-41 and the ATM gene.

[0156] In this work we demonstrate that the correct sequence of the rad3gene places its product in the family of protein/lipid kinases relatedto ATM. Over-expression of kinase-defective rad3 mutant in S. pombecauses a dominant negative phenotype, which suggests that Rad3 is actingas a member of a protein complex whose inter is necessary for checkpointOnion. This is consistent with the observation that rad1, rad9, rad17,rad26 and has1 deletion mutants all have phenotype indistinguishablefrom rad3.d (Sheldrick and Carr, 1993). Unexpectedly, unlike theremaining checkpoint rad genes, high level over-expression of eitherwild type or mutant rad alleles inhibits cell growth and causes mitosisto occur at a reduced cell size, indicative of premature entry intomitosis. This “semi wee” phenotype is not observed in the null mutant,and may indicate interference in a second pathway whose functionoverlaps with that of Rad3 and acts to inhibit mitosis. A candidate forsuch a pathway is the ATM/TEL1 pathway which has been shown to have someoverlapping functions with the ESR1(MEC1/SAD1) pathway (Morrow et al..1995).

[0157] The structure of ATM is most closely related to the Te11p, whichis involved in maintaining telomere length (Greenwell et al., 1995).However, ATM function also appears related to that of theRad3/Esr1p/mei-41 products. Following the initial discovery of the ATMgene and its sequence relationship to the rad3/ESR1 genes and to TEL1,it was not clear whether, as in many cases in yeast, the gene hadduplicated and diverged in yeast, or whether the two yeast proteinsdefined conserved sub-families of closely related genes. The significantfinding of this work is the identification of a human gene, ATR, whichis more closely related to rad3/ESR1/mei-41. his defines two sullydistinct checkpoint related subfamilies of protein/lipid kinases thatare conserved throughout eukaryotic evolution. Although the proteins inthese two subfamilies may have some overlapping functions, they probablycontrol different processes. For example: the rad3 sub-family in yeastcontrol all the G1 and G2 DNA damage checkpoints in response to both uvand ionising radiation, and the S phase checkpoint which preventsmitosis following inhibition of replication (Al-Khodairy and Carr, 1992:Allen et al., 1994; Weinert et al., 1994). In contrast, A-T cells haveabnormal responses to a narrow range of DNA damaging agents includingionising radiation, biomycin and neocarzinostatin, which produce strandbreaks in DNA as a consequence of radical attack. The response to uv andmost chemical carcinogens is normal, as is the response to theinhibition of DNA synthesis. It is possible that some or all of theremaining DNA damage checkpoints and the S phase checkpoint arecontrolled by ATR.

[0158] Experimental procedures

[0159] Strains, plasmids and media

[0160] Standard genetic techniques, growth conditions and media for S.pombe are described in Gutz et al. (1974). S. pombe strain sp011(ura4.D18, leu.1.32 ade6.704 h⁻) has been described previously (Murrayet al. 1992). Plasmid pSUB41 was a gift from S. Sub i (Seaton et al.1992).

[0161] Cloning of S. pombe rad3

[0162] A 4.0 kb Kpn1 fragment was excised from pSUB41 and sequenced inboth directions to obtain the 5′ rad3 sequence. The 3′ clone wasidentified from a genomic library (Barbet et al. 1992) by colonyhybridisation using a 1 kb 3′ probe derived from the published rad3sequence, and sequenced in both directions. In this way, the sequence ofthe entire rad3 gene was assembled.

[0163] Null and “kinase dead” rad3 mutants

[0164] A construct of rad, in which the 794 amino acids between aa 1477and aa2271 (including the kinase domain) were replaced with a ura4+gene, was created using the A methodology described in Barbet et al.(1992). A linear fragment of this was used to transform spoil to uracilprototropy and single copy integration at the rad3 locus was checked bySouthern blotting. To create the site specific kinase null mutations, aC-terminal 3.01 kb BamHI fragment of rad3 was mutated with either (A:GTTTTCGCCATGGCGCGCTCCCAAACCCAA, B: TTCATCAAACAATATCTTTTCGCCATGGCG, or C;CAAAAGACAGTTGAATTCGACATGGATAG) in order to introduce either the D2230A,N2235K or D2249E mutations into the kinase domain. Analogous changeshave previously been used in the analysis of P13 kinase VPS34 of S.cerevisiae (Schu et al. 1993). These fragments were then used totransform the rad3id null mutant and gene replacements selected by theirability to grow on FOA containing media (Grimm et al. 1988). All strainswere checked by Southern blotting. Full length expression constructs ofrad3.D2230A were created in pREP1 and pREP41 (Maundrell, 1990) bystandard subcloning following introduction of an NdeI site at the ATGand deletion of three internal NdeI sites.

[0165] UV radiation sensitivity strip tests

[0166] Expression from REP1 (high) and REP41 (intermediate) was inducedby the absence of thiamine for 18 hours prior to plating. Plates wereirradiated with a gradient of uv doses down the plate from 0 to 300 Jm⁻²according to the settings on a Stratagene Stratalinker.

[0167] Cloning and expression of ATR

[0168] To isolate an appropriate probe for identifying cDNAscorresponding to a human rad3 homologue, degenerate oligonucleotideswere designed against the amino acids LGLGDRH (5′ oligo; oDH18) andHVDF[D/N]C (3′ oligo; oDH-16) of Rad3/Esr1p. Inosine was incorporated atpositions of four-fold degeneracy, and primers were tailed with BamHI(oDH18) and EcoRI (oDH16) to facilitate cloning. DNA sequence analysisof the ˜100 bp PCR product obtained from amplification of peripheralblood leukocyte cDNA demonstrated significant similarity to MEC1/rad3.This sequence was used to synthesize a non-degenerate primer (oDH-23;GACGCAGAATTCACCAGTCAAAGAATCAAAGAG) for PCR with an additional degenerateprimer (oDH17) designed against he amino acid sequenceKFPP[I/V][L/F]Y[Q/E]WF of Rad3/Esr1p. The 174 bp product of thisreaction was used directly to screen a macrophage cDNA library. Fourpositive clones were isolated (the lest approximately 3 kb).

[0169] In parallel, database searches with full length S pombe rad3derived from the EMBL database a human cDNA clone, HSAAADPDG, as apotential homologue of rad3, if a single frameshift was allowed for inthe 233 bp sequence. This 233 bp sequence is contained within a 1.6 kbclone obtained from Dr. N. Affara, Human Molecular Genetics ResearchGroup, Cambridge University, UK. The entire clone (1.6 kb) was sequencedand lies within the cDNA clones identified by degenerate PCR and libraryscreens. To identify the whole gene, RACE PCR experiments were performedon cDNA derived from placental and thymus mRNA using the instructionsprovided with a Clontech Marathon Kit. Gene specific primers werederived from the cDNA clones. From these experiments, a 8239 bp cDNAsequence was assembled with an internal ORF of 2644 amino acids, a 79 bp5′ noncoding region, a 194 bp 3′ noncoding region and a poly A⁻ tail.Parts of the sequence were determined solely by PCR. To avoid errors,clones from a minimum of 3 independent PCR reactions were sequenced inboth directions.

[0170] The 233 bp sequence corresponds to the sequence of nucleotides6809 to 7042 (234 nt in total) of Seq. ID No. 1 except for a single basedeletion at position 6942. This sequence encodes amino acids 2244 to2320 of Seq. ID No. 2.

[0171] The sequence of the “1.6 kb” insert corresponds to nucleotides5725 to 7104 (1353 nt) of Seq. ID No. 1, and encodes amino acids 1892 to2340 of Seq. ID No. 2.

[0172] Northern blot hybridisation: a 1.3 kb PCR product was amplifiedin the presence of ³²P-dCTP using primers 279-3 (TGGATGATGACAGCTGTGTC)and 279-6 (TGTAGTCGCTGCTCAATGTC). A nylon membrane containing 2 μg ofsize-fractionated polyA+RNA from a variety of human tissue sources(Clontech Laboratories) was probed as recommended by the manufacturerexcept that the final wash was performed at 55° C. rather than 50° C. tominimize the possibility of cross-hybridisation to related sequences.

[0173] Mapping ATR.

[0174] We mapped the ATR gene to chromosome 3 by a combination offluorescent in situ hybridisation and polymerase chain reaction (PCR)based assays. FISH analysis using a cDNA clone identified the AIR geneon chromosome 3, at approximately position q22-23. PCR analysis alsoidentified ATR on chromosome 3. Two primers (oATR23:GACGCAGAATTCACCAGTCAAAGAATCAAAGAG and oATR26: TGGTTTCTGAGAACATTCCCTGA)which amplify a 257 bp fragment of the ATR gene were used on DNA derivedfrom human/rodent somatic cell hybrids containing various humanchromosome panels available from the NIGMS Human Genetic Mutant CellRepository (Drwinga et al. 1993). PCR with the same primers was used tosub-localise ATR to a specific region on chromosome 3. The templates forthese amplifications consisted of DNA samples from patients withtruncations along chromosome 3 (Leach et al. 1994).

[0175] Immunoprecipitation (IP) and kinase assays with Rad3

[0176] The S. pombe rad3 and human ATR genes were cloned into pREP41expression vector for complementation studies. To -tag the proteins,versions of these vectors containing in-frame N terminal tag sequences,either a double myc or a triple HA tag, were used (Griffiths et al.1995). Tagged proteins were expressed by growing in media withoutthiamine (Maundrell. 1990). Yeast cells lysed in lysis buffer (25 mMTris.Cl pH 7.5. 60 mM B-glycerophosphate, 0.1 mM Na₃VO₄, 1% TritonX-100, 50 mM MaCl, 2 mM EDTA. 50 mM NaF, 1 mM phenylmethylsulfonylfluoride [PMSF], 5 μg/ml leupeptin, 5 μg/ml aprotinin, 1 mM DTT) by theaddition of glass beads followed by treatment in a dismembrinator for 2minutes. For IP's 300 μg total protein extract was incubated on ice withthe appropriate antibody for 30 min and the immune complexesprecipitated by mixing with Protein G beads for a further 30 min at 4°C. For kinase assays, the immune complexes were washed 4 timed withLysis buffer, once with Kinase Buffer (25 mM Hepes pH7.7; 50 mM KCl; 10mM MgCl₂; 0.1% NP-40; 2% glycerol; 1 mM DTT), and incubated in KinaseBuffer with 10 μM ATP [50 Ci/mmol]) for 15 minutes at 30° C. Thereactions were stopped with 20 ul 2×SDS sample buffer prior toseparation on 6% polyacrylamide gels. Rad3 IP's contained severalphosphorylated products, including one which comigrated with Rad3protein itself on Western analysis.

[0177] References

[0178] Al-Khodairy, F., and. Carr, A. M. (1992). DNA repair mutantsdefining G2 checkpoint pathways in Schizosaccharomyces pombe. EMBO J.11, 1343-1350.

[0179] Al-Khodairy, F., Fotou, E., Sheldrick, K. S., Griffiths, D. J.F., Lehmann A. R. and Carr, A. M. (1994). Identification andcharacterization of new elements involved in checkpoints and feedbackcontrols in fission yeast. Mol. Biol. Cell 5, 147-160.

[0180] Allen, J. B., Zhou, Z., Siede, W., Friedberg, E. C. and Elledge,S. J. (1994) The SAD1/RAD53 protein kinase controls multiple checkpointsand DNA damage-induced transcription in yeast. Genes Dev. 8, 2416-2428.

[0181] Barbet, N. C., Muriel, W. J., and Carr, A. M. (1992) Versatileshuttle vectors and genomic libraries for use with Schizosaccharomycespombe, Gene 114, 59-66.

[0182] Beamish, H. and Lavin, M. F. (1994) Radiosensitivity inataxia-telangiectasia: anomalies in radiation-induced cell cycle delay.Int. J. Radiat Biol. 65, 175-184.

[0183] Carr, A. M. and Hoekstra, M. F. (1995) The Cellular Responses toDNA Damage. Trends in Cell Biology 5, 32-40.

[0184] Chien et al., (1991) Proc. Natl. Acad Sci USA 88, 9578-9582

[0185] Deng, C., Zhang, P., Harper, J. W., Elledge, S. J. and Leder, P.J. (1995) Mice lacking p21^(CP1/WAF1) undergo normal development, butare defective in G1 checkpoint control. Cell, (in press).

[0186] Drwinga, H. L., Tojia, L. H., Kim, C. H., Greene, A. E., andMulovor, R. A. (1993). NIGMS Human/Rodent Somatic Cell Hybrid MappingPanels 1 and 2. Genomics 16:311-314.

[0187] El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B.,Parson, R., Trent, I. M., Lin D., Mercer, W. E., Kinzler, K. W. andVogelstein, B. (1993) WAF1, a potential mediator of p53 tumoursuppression. Cell 75, 817-825

[0188] Enoch, T., Carr, A. M. and Nurse, P. (1992). Fission yeast genesinvolved in coupling mitosis to completion of DNA-replication. GenesDev. 6, 2035-2046.

[0189] Greenwell, P. W., Kronmal, S. L., Porter, S. E., Gassenhuber, J.,Obermaier. B. and Petes, T. D. (1995) TEL1, a gene involved incontrolling telomere length in Saccharomyces cerevisiae, is homologousto the human ataxia telangiectasia (ATM) gene. Cell submitted.

[0190] Grimm C., Kholi, J. Murray, J. M. and Maundrell, K. (1988)Genetic engineering of Schizosaccharomyces pombe: a system for genedisruption and replacement using the ura4 gene as a selectable marker.Mol. Gen. Genet. 215, 81-86.

[0191] Gutz, H., Heslot, H. Leupold, U. and Loprieno, N. (1974). In“Handbook of Genetics”, King R. C., Ed., Plenum Press, New York, Vol. 1,395-446.

[0192] Hari, K. L., Santerre. A.. Sekelsky, J. J., McKim, K. S., Boyd,J. B. and Hawley, R. S. (1995) The mei-4 1 gene of Drosophilamelanogaster is functionally homologous to the human ataxia telangiec

[0193] Harper, J. W., Adami, G., Wei, N., Keyomarsi, K. and Elledge, S.J. (1993) The 21 kD Cdk interacting protein Cip1 is a potent inhibitorof G1 cyclin dependent kinases. Cell 75, 805-816.

[0194] Hartwell, L. H., and Kastan, M. B. (1994). Cell cycle control andCancer.,Science 266, 1821-1828.

[0195] Hiles, I. D., Otsu, M., Volinia, S., Fry, M. J., Gout, I., Dhand,R., Panayotou, G., Ruiz-Larrea, F., Thompson, A., Totty, N. F., Hsuan,J. J., Courtneidge, S. A., Parker, P. J. and Waterfield M. D. (1992)Phosphatidylinositol 3-kinase: structure and expression of the 110 kdcatalytic subunit. Cell 70, 419-429.

[0196] Jimenez, G., Yucel, J., Rowley, R. and Subramani S. (1992) Therad3+ gene of Schizosaccharomyces pombe is involved in multiplecheckpoint functions and in DNA repair. Proc Natl. Acad. Sci. USA 87,4952-4956

[0197] Kato, R. and Ogawa, H. (1994) An essential gene, ESR1, isrequired for mitotic cell growth, DNA repair and Meiotic recombinationin Saccharomyces cerevisiae. Nucleic Acids Res. 22, 3104-3112.

[0198] Lamb, J. R., Petit-Frere, C., Broughton, B. C., Lehmann, A. R.and Green, M. H. L. (1989) Inhibition of DNA replication by ionizingradiation is mediated by a trans acting factor. Int. J. Radiat. Biol.56, 125-130.

[0199] Leach, R. J., Chinn, R., Reus, B. E., Hayes, S., Schantz, L.,Dubois, B., Overhauser, J., Ballabio, A., Drabkin, H.. Lewis, B. T.,Mendgen, G., and Naylor, S. L. (1994) Regional Localisation of 188Sequence Tagged Sites on a Somatic Cell Hybrid Mapping Panel for HumanChromosome 3 Genomics 24, 549-556

[0200] Maundrell, K. (1990), nmt1 of fission yeast. A highly transcribedgene completely repressed by thiamine. J. Biol. Chem. 265, 10857-10864.

[0201] Morrow. D. M.. Tagle, D. A.. Shiloh, Y., Collins F. S. andHieter, P. (1995) HAT1/TEL1, a Saccharomyces cerevisiae homologue of thehuman gene mutated in ataxia-telangiectasia, is functionally related tothe yeast checkpoint gene MEC1/ESR1. Cell submitted.

[0202] Murray, J. M., Doe, C. Schenk, P. Carr, A. M.. Lehmann, A. R andWatts, F. Z. (1992) Cloning and characterization of the S. pombe rad15gene, a homologue to the S. cerevisiae RAD3 and human ERCC2 genesNucleic Acids Res. 20, 2673-2678.

[0203] Nasim, A, and Smith, B. P. (1975) Genetic control of radiationsensitivity in Schizosaccharomyces pombe. Genetics 79, 573-582.

[0204] Painter, R. B. and Young, B. R (1980) Radiosensitivity inataxia-telangiectasia: A new explanation. Proc. Natl. Acad. Sci. USA.77, 7315-7317

[0205] Rowley, R. Subramani, S. and Young, P. G. (1992). Checkpointcontrols in Schizosaccharomyces pombe, rad1. EMBO J. 11, 1335-1342.

[0206] Savitsky, K., Bar-Shira, A., Gilad, S., Rotman, G., Ziv, Y..Vanagaite L., Tagle, D. A., Smith, S., Uziel, T., Sfez, S., Ashkenazi,M, Pecker, I., Frydman, M., Harnik, R., Patanjali, S. R., Simmons, A.,Clines, G. A., Sartiel, A., Gatti, R. A., Chessa, L., Sanal, O., Lavine,M. F., Jaspers, N. G. J., Taylor, M. R, Arlett, C. F., Miki, T.,Weissman, S. M., Lovett, M., Collins, F. S. and Shiloh, Y. (1995). Asingle ataxia telangiectasia gene with a product similar to PI-3 kinase.Science 286, 1749-1753.

[0207] Seaton, B. L., Yucel, I., Sunnerhagen P. and Subramani, S.(1992). Isolation and characterisation of the Schizosaccharomyces pomberad3 gene which is involved in the DNA damage and DNA synthesischeckpoints. Gene 119, 83-89.

[0208] Schu, P. V., Takegawa, K., Fry, M. J., Stack, J. H., Waterfield,M. D. and Emr, S. D. (1993) Phosphatidylinositol 3-kinase encoded byyeast VPS34 gene essential for protein sorting. Science 260, 88-91.

[0209] Sheldrick K. S. and Carr. A. N (1993). Feedback controls and G2checkpoints. fission yeast as a model system. BioEssays 15, 775-782.

[0210] Walworth, N., Davey, S. and Beach, D. (1993). Fission yeast chk1protein kinase link the rad checkpoint pathway to cdc2. Nature 363,368-371.

[0211] Weinert, T. A., and Hartwell, L. H. (1988). The RAD9 genecontrols the cell cycle response to DNA damage in Saccharomycescerevisiae. Science 241, 317-322.

[0212] Weinert, T. A., Kiser, G. L. Hartwell, L. H. (1994). Mitoticcheckpoint genes in budding yeast and the dependence of mitosis on DNAreplication and repair. Genes Dev. 8, 652-665.

1 14 1 8239 DNA Homo sapiens CDS (80)..(8011) 1 gcgctcttcc ggcagcggtacgtttggaga cgccgggaac ccgcgttggc gtggttgact 60 agtgcctcgc agcctcagc atgggg gaa cat ggc ctg gag ctg gct tcc atg 112 Met Gly Glu His Gly Leu GluLeu Ala Ser Met 1 5 10 atc ccc gcc ctg cgg gag ctg ggc agt gcc aca ccagag gaa tat aat 160 Ile Pro Ala Leu Arg Glu Leu Gly Ser Ala Thr Pro GluGlu Tyr Asn 15 20 25 aca gtt gta cag aag cca aga caa att ctg tgt caa ttcatt gac cgg 208 Thr Val Val Gln Lys Pro Arg Gln Ile Leu Cys Gln Phe IleAsp Arg 30 35 40 ata ctt aca gat gta aat gtt gtt gct gta gaa ctt gta aagaaa act 256 Ile Leu Thr Asp Val Asn Val Val Ala Val Glu Leu Val Lys LysThr 45 50 55 gac tct cag cca acc tcc gtg atg ttg ctt gat ttc atc cag catatc 304 Asp Ser Gln Pro Thr Ser Val Met Leu Leu Asp Phe Ile Gln His Ile60 65 70 75 atg aaa tcc tcc cca ctt atg ttt gta aat gtg agt gga agc catgag 352 Met Lys Ser Ser Pro Leu Met Phe Val Asn Val Ser Gly Ser His Glu80 85 90 cgc aaa ggc agt tgt att gaa ttc agt aat tgg atc ata acg aga ctt400 Arg Lys Gly Ser Cys Ile Glu Phe Ser Asn Trp Ile Ile Thr Arg Leu 95100 105 ctg cgg att gca gca act ccc tcc tgt cat ttg tta cac aag aaa atc448 Leu Arg Ile Ala Ala Thr Pro Ser Cys His Leu Leu His Lys Lys Ile 110115 120 tgt gaa gtc atc tgt tca tta tta ttt ctt ttt aaa agc aag agt cct496 Cys Glu Val Ile Cys Ser Leu Leu Phe Leu Phe Lys Ser Lys Ser Pro 125130 135 gct att ttt ggg gta ctc aca aaa gaa tta tta caa ctt ttt gaa gac544 Ala Ile Phe Gly Val Leu Thr Lys Glu Leu Leu Gln Leu Phe Glu Asp 140145 150 155 ttg gtt tac ctc cat aga aga aat gtg atg ggt cat gct gtg gaatgg 592 Leu Val Tyr Leu His Arg Arg Asn Val Met Gly His Ala Val Glu Trp160 165 170 cca gtg gtc atg agc cga ttt tta agt caa tta gat gaa cac atggga 640 Pro Val Val Met Ser Arg Phe Leu Ser Gln Leu Asp Glu His Met Gly175 180 185 tat tta caa tca gct cct ttg cag ttg atg agt atg caa aat ttagaa 688 Tyr Leu Gln Ser Ala Pro Leu Gln Leu Met Ser Met Gln Asn Leu Glu190 195 200 ttt att gaa gtc act tta tta atg gtt ctt act cgt att att gcaatt 736 Phe Ile Glu Val Thr Leu Leu Met Val Leu Thr Arg Ile Ile Ala Ile205 210 215 gtg ttt ttt aga agg caa gaa ctc tta ctt tgg cag ata ggt tgtgtt 784 Val Phe Phe Arg Arg Gln Glu Leu Leu Leu Trp Gln Ile Gly Cys Val220 225 230 235 ctg cta gag tat ggt agt cca aaa att aaa tcc cta gca attagc ttt 832 Leu Leu Glu Tyr Gly Ser Pro Lys Ile Lys Ser Leu Ala Ile SerPhe 240 245 250 tta aca gaa ctt ttt cag ctt gga gga cta cca gca caa ccagct agc 880 Leu Thr Glu Leu Phe Gln Leu Gly Gly Leu Pro Ala Gln Pro AlaSer 255 260 265 act ttt ttc agc tca ttt ttg gaa tta tta aaa cac ctt gtagaa atg 928 Thr Phe Phe Ser Ser Phe Leu Glu Leu Leu Lys His Leu Val GluMet 270 275 280 gat act gac caa ttg aaa ctc tat gaa gag cca tta tca aagctg ata 976 Asp Thr Asp Gln Leu Lys Leu Tyr Glu Glu Pro Leu Ser Lys LeuIle 285 290 295 aag aca cta ttt ccc ttt gaa gca gaa gct tat aga aat attgaa cct 1024 Lys Thr Leu Phe Pro Phe Glu Ala Glu Ala Tyr Arg Asn Ile GluPro 300 305 310 315 gtc tat tta aat atg ctg ctg gaa aaa ctc tgt gtc atgttt gaa gac 1072 Val Tyr Leu Asn Met Leu Leu Glu Lys Leu Cys Val Met PheGlu Asp 320 325 330 ggt gtg ctc atg cgg ctt aag tct gat ttg cta aaa gcagct ttg tgc 1120 Gly Val Leu Met Arg Leu Lys Ser Asp Leu Leu Lys Ala AlaLeu Cys 335 340 345 cat tta ctg cag tat ttc ctt aaa ttt gtg cca gct gggtat gaa tct 1168 His Leu Leu Gln Tyr Phe Leu Lys Phe Val Pro Ala Gly TyrGlu Ser 350 355 360 gct tta caa gtc agg aag gtc tat gtg aga aat att tgtaaa gct ctt 1216 Ala Leu Gln Val Arg Lys Val Tyr Val Arg Asn Ile Cys LysAla Leu 365 370 375 ttg gat gtg ctt gga att gag gta gat gca gag tac ttgttg ggc cca 1264 Leu Asp Val Leu Gly Ile Glu Val Asp Ala Glu Tyr Leu LeuGly Pro 380 385 390 395 ctt tat gca gct ttg aaa atg gaa agt atg gaa atcatt gag gag att 1312 Leu Tyr Ala Ala Leu Lys Met Glu Ser Met Glu Ile IleGlu Glu Ile 400 405 410 caa tgc caa act caa cag gaa aac ctc agc agt aatagt gat gga ata 1360 Gln Cys Gln Thr Gln Gln Glu Asn Leu Ser Ser Asn SerAsp Gly Ile 415 420 425 tca ccc aaa agg cgt cgt ctc agc tcg tct cta aaccct tct aaa aga 1408 Ser Pro Lys Arg Arg Arg Leu Ser Ser Ser Leu Asn ProSer Lys Arg 430 435 440 gca cca aaa cag act gag gaa att aaa cat gtg gacatg aac caa aag 1456 Ala Pro Lys Gln Thr Glu Glu Ile Lys His Val Asp MetAsn Gln Lys 445 450 455 agc ata tta tgg agt gca ctg aaa cag aaa gct gaatcc ctt cag att 1504 Ser Ile Leu Trp Ser Ala Leu Lys Gln Lys Ala Glu SerLeu Gln Ile 460 465 470 475 tcc ctt gaa tac agt ggc cta aag aat cct gttatt gag atg tta gaa 1552 Ser Leu Glu Tyr Ser Gly Leu Lys Asn Pro Val IleGlu Met Leu Glu 480 485 490 gga att gct gtt gtc tta caa ctg act gct ctgtgt act gtt cat tgt 1600 Gly Ile Ala Val Val Leu Gln Leu Thr Ala Leu CysThr Val His Cys 495 500 505 tct cat caa aac atg aac tgc cgt act ttc aaggac tgt caa cat aaa 1648 Ser His Gln Asn Met Asn Cys Arg Thr Phe Lys AspCys Gln His Lys 510 515 520 tcc aag aag aaa cct tct gta gtg ata act tggatg tca ttg gat ttt 1696 Ser Lys Lys Lys Pro Ser Val Val Ile Thr Trp MetSer Leu Asp Phe 525 530 535 tac aca aaa gtg ctt aag agc tgt aga agt ttgtta gaa tct gtt cag 1744 Tyr Thr Lys Val Leu Lys Ser Cys Arg Ser Leu LeuGlu Ser Val Gln 540 545 550 555 aaa ctg gac ctg gag gca acc att gat aaggtg gtg aaa att tat gat 1792 Lys Leu Asp Leu Glu Ala Thr Ile Asp Lys ValVal Lys Ile Tyr Asp 560 565 570 gct ttg att tat atg caa gta aac agt tcattt gaa gat cat atc ctg 1840 Ala Leu Ile Tyr Met Gln Val Asn Ser Ser PheGlu Asp His Ile Leu 575 580 585 gaa gat tta tgt ggt atg ctc tca ctt ccatgg att tat tcc cat tct 1888 Glu Asp Leu Cys Gly Met Leu Ser Leu Pro TrpIle Tyr Ser His Ser 590 595 600 gat gat ggc tgt tta aag ttg acc aca tttgcc gct aat ctt cta aca 1936 Asp Asp Gly Cys Leu Lys Leu Thr Thr Phe AlaAla Asn Leu Leu Thr 605 610 615 tta agc tgt agg att tca gat agc tat tcacca cag gca caa tca cga 1984 Leu Ser Cys Arg Ile Ser Asp Ser Tyr Ser ProGln Ala Gln Ser Arg 620 625 630 635 tgt gtg ttt ctt ctg act ctg ttt ccaaga aga ata ttc ctt gag tgg 2032 Cys Val Phe Leu Leu Thr Leu Phe Pro ArgArg Ile Phe Leu Glu Trp 640 645 650 aga aca gca gtt tac aac tgg gcc ctgcag agc tcc cat gaa gta atc 2080 Arg Thr Ala Val Tyr Asn Trp Ala Leu GlnSer Ser His Glu Val Ile 655 660 665 cgg gct agt tgt gtt agt gga ttt tttatc tta ttg cag cag cag aat 2128 Arg Ala Ser Cys Val Ser Gly Phe Phe IleLeu Leu Gln Gln Gln Asn 670 675 680 tct tgt aac aga gtt ccc aag att cttata gat aaa gtc aaa gat gat 2176 Ser Cys Asn Arg Val Pro Lys Ile Leu IleAsp Lys Val Lys Asp Asp 685 690 695 tct gac att gtc aag aaa gaa ttt gcttct ata ctt ggt caa ctt gtc 2224 Ser Asp Ile Val Lys Lys Glu Phe Ala SerIle Leu Gly Gln Leu Val 700 705 710 715 tgt act ctt cac ggc atg ttt tatctg aca agt tct tta aca gaa cct 2272 Cys Thr Leu His Gly Met Phe Tyr LeuThr Ser Ser Leu Thr Glu Pro 720 725 730 ttc tct gaa cac gga cat gtg gacctc ttc tgt agg aac ttg aaa gcc 2320 Phe Ser Glu His Gly His Val Asp LeuPhe Cys Arg Asn Leu Lys Ala 735 740 745 act tct caa cat gaa tgt tca tcttct caa cta aaa gct tct gtc tgc 2368 Thr Ser Gln His Glu Cys Ser Ser SerGln Leu Lys Ala Ser Val Cys 750 755 760 aag cca ttc ctt ttc cta ctg aaaaaa aaa ata cct agt cca gta aaa 2416 Lys Pro Phe Leu Phe Leu Leu Lys LysLys Ile Pro Ser Pro Val Lys 765 770 775 ctt gct ttc ata gat aat cta catcat ctt tgt aag cat ctt gat ttt 2464 Leu Ala Phe Ile Asp Asn Leu His HisLeu Cys Lys His Leu Asp Phe 780 785 790 795 aga gaa gat gaa aca gat gtaaaa gca gtt ctt gga act tta tta aat 2512 Arg Glu Asp Glu Thr Asp Val LysAla Val Leu Gly Thr Leu Leu Asn 800 805 810 tta atg gaa gat cca gac aaagat gtt aga gtg gct ttt agt gga aat 2560 Leu Met Glu Asp Pro Asp Lys AspVal Arg Val Ala Phe Ser Gly Asn 815 820 825 atc aag cac ata ttg gaa tccttg gac tct gaa gat gga ttt ata aag 2608 Ile Lys His Ile Leu Glu Ser LeuAsp Ser Glu Asp Gly Phe Ile Lys 830 835 840 gag ctt ttt gtc tta aga atgaag gaa gca tat aca cat gcc caa ata 2656 Glu Leu Phe Val Leu Arg Met LysGlu Ala Tyr Thr His Ala Gln Ile 845 850 855 tca aga aat aat gag ctg aaggat acc ttg att ctt aca aca ggg gat 2704 Ser Arg Asn Asn Glu Leu Lys AspThr Leu Ile Leu Thr Thr Gly Asp 860 865 870 875 att gga agg gcc gca aaagga gat ttg gta cca ttt gca ctc tta cac 2752 Ile Gly Arg Ala Ala Lys GlyAsp Leu Val Pro Phe Ala Leu Leu His 880 885 890 tta ttg cat tgt ttg ttatcc aag tca gca tct gtc tct gga gca gca 2800 Leu Leu His Cys Leu Leu SerLys Ser Ala Ser Val Ser Gly Ala Ala 895 900 905 tac aca gaa att aga gctctg gtt gca gct aaa agt gtt aaa ctg caa 2848 Tyr Thr Glu Ile Arg Ala LeuVal Ala Ala Lys Ser Val Lys Leu Gln 910 915 920 agt ttt ttc agc cag tataag aaa ccc atc tgt cag ttt ttg gta gaa 2896 Ser Phe Phe Ser Gln Tyr LysLys Pro Ile Cys Gln Phe Leu Val Glu 925 930 935 tcc ctt cac tct agt cagatg aca gca ctt ccg aat act cca tgc cag 2944 Ser Leu His Ser Ser Gln MetThr Ala Leu Pro Asn Thr Pro Cys Gln 940 945 950 955 aat gct gac gtg cgaaaa caa gat gtg gct cac cag aga gaa atg gct 2992 Asn Ala Asp Val Arg LysGln Asp Val Ala His Gln Arg Glu Met Ala 960 965 970 tta aat acg ttg tctgaa att gcc aac gtt ttc gac ttt cct gat ctt 3040 Leu Asn Thr Leu Ser GluIle Ala Asn Val Phe Asp Phe Pro Asp Leu 975 980 985 aat cgt ttt ctt actagg aca tta caa gtt cta cta cct gat ctt gct 3088 Asn Arg Phe Leu Thr ArgThr Leu Gln Val Leu Leu Pro Asp Leu Ala 990 995 1000 gcc aaa gca agc cctgca gct tct gct ctc att cga act tta gga aaa 3136 Ala Lys Ala Ser Pro AlaAla Ser Ala Leu Ile Arg Thr Leu Gly Lys 1005 1010 1015 caa tta aat gtcaat cgt aga gag att tta ata aac aac ttc aaa tat 3184 Gln Leu Asn Val AsnArg Arg Glu Ile Leu Ile Asn Asn Phe Lys Tyr 1020 1025 1030 1035 att ttttct cat ttg gtc tgt tct tgt tcc aaa gat gaa tta gaa cgt 3232 Ile Phe SerHis Leu Val Cys Ser Cys Ser Lys Asp Glu Leu Glu Arg 1040 1045 1050 gccctt cat tat ctg aag aat gaa aca gaa att gaa ctg ggg agc ctg 3280 Ala LeuHis Tyr Leu Lys Asn Glu Thr Glu Ile Glu Leu Gly Ser Leu 1055 1060 1065ttg aga caa gat ttc caa gga ttg cat aat gaa tta ttg ctg cgt att 3328 LeuArg Gln Asp Phe Gln Gly Leu His Asn Glu Leu Leu Leu Arg Ile 1070 10751080 gga gaa cac tat caa cag gtt ttt aat ggt ttg tca ata ctt gcc tca3376 Gly Glu His Tyr Gln Gln Val Phe Asn Gly Leu Ser Ile Leu Ala Ser1085 1090 1095 ttt gca tcc agt gat gat cca tat cag ggc ccg aga gat atcata tca 3424 Phe Ala Ser Ser Asp Asp Pro Tyr Gln Gly Pro Arg Asp Ile IleSer 1100 1105 1110 1115 cct gaa ctg atg gct gat tat tta caa ccc aaa ttgttg ggc att ttg 3472 Pro Glu Leu Met Ala Asp Tyr Leu Gln Pro Lys Leu LeuGly Ile Leu 1120 1125 1130 gct ttt ttt aac atg cag tta ctg agc tct agtgtt ggc att gaa gat 3520 Ala Phe Phe Asn Met Gln Leu Leu Ser Ser Ser ValGly Ile Glu Asp 1135 1140 1145 aag aaa atg gcc ttg aac agt ttg atg tctttg atg aag tta atg gga 3568 Lys Lys Met Ala Leu Asn Ser Leu Met Ser LeuMet Lys Leu Met Gly 1150 1155 1160 ccc aaa cat gtc agt tct gtg agg gtgaag atg atg acc aca ctg aga 3616 Pro Lys His Val Ser Ser Val Arg Val LysMet Met Thr Thr Leu Arg 1165 1170 1175 act ggc ctt cga ttc aag gat gatttt cct gaa ttg tgt tgc aga gct 3664 Thr Gly Leu Arg Phe Lys Asp Asp PhePro Glu Leu Cys Cys Arg Ala 1180 1185 1190 1195 tgg gac tgc ttt gtt cgctgc ctg gat cat gct tgt ctg ggc tcc ctt 3712 Trp Asp Cys Phe Val Arg CysLeu Asp His Ala Cys Leu Gly Ser Leu 1200 1205 1210 ctc agt cat gta atagta gct ttg tta cct ctt ata cac atc cag cct 3760 Leu Ser His Val Ile ValAla Leu Leu Pro Leu Ile His Ile Gln Pro 1215 1220 1225 aaa gaa act gcagct atc ttc cac tac ctc ata att gaa aac agg gat 3808 Lys Glu Thr Ala AlaIle Phe His Tyr Leu Ile Ile Glu Asn Arg Asp 1230 1235 1240 gct gtg caagat ttt ctt cat gaa ata tat ttt tta cct gat cat cca 3856 Ala Val Gln AspPhe Leu His Glu Ile Tyr Phe Leu Pro Asp His Pro 1245 1250 1255 gaa ttaaaa aag ata aaa gcc gtt ctc cag gaa tac aga aag gag acc 3904 Glu Leu LysLys Ile Lys Ala Val Leu Gln Glu Tyr Arg Lys Glu Thr 1260 1265 1270 1275tct gag agc act gat ctt cag aca act ctt cag ctc tct atg aag gcc 3952 SerGlu Ser Thr Asp Leu Gln Thr Thr Leu Gln Leu Ser Met Lys Ala 1280 12851290 att caa cat gaa aat gtc gat gtt cgt att cat gct ctt aca agc ttg4000 Ile Gln His Glu Asn Val Asp Val Arg Ile His Ala Leu Thr Ser Leu1295 1300 1305 aag gaa acc ttg tat aaa aat cag gaa aaa ctg ata aag tatgca aca 4048 Lys Glu Thr Leu Tyr Lys Asn Gln Glu Lys Leu Ile Lys Tyr AlaThr 1310 1315 1320 gac agt gaa aca gta gaa cct att atc tca cag ttg gtgaca gtg ctt 4096 Asp Ser Glu Thr Val Glu Pro Ile Ile Ser Gln Leu Val ThrVal Leu 1325 1330 1335 ttg aaa ggt tgc caa gat gca aac tct caa gct cggttg ctc tgt ggg 4144 Leu Lys Gly Cys Gln Asp Ala Asn Ser Gln Ala Arg LeuLeu Cys Gly 1340 1345 1350 1355 gaa tgt tta ggg gaa ttg ggg gcg ata gatcca ggt cga tta gat ttc 4192 Glu Cys Leu Gly Glu Leu Gly Ala Ile Asp ProGly Arg Leu Asp Phe 1360 1365 1370 tca aca act gaa act caa gga aaa gatttt aca ttt gtg act gga gta 4240 Ser Thr Thr Glu Thr Gln Gly Lys Asp PheThr Phe Val Thr Gly Val 1375 1380 1385 gaa gat tca agc ttt gcc tat ggatta ttg atg gag cta aca aga gct 4288 Glu Asp Ser Ser Phe Ala Tyr Gly LeuLeu Met Glu Leu Thr Arg Ala 1390 1395 1400 tac ctt gcg tat gct gat aatagc cga gct caa gat tca gct gcc tat 4336 Tyr Leu Ala Tyr Ala Asp Asn SerArg Ala Gln Asp Ser Ala Ala Tyr 1405 1410 1415 gcc att cag gag ttg ctttct att tat gac tgt aga gag atg gag acc 4384 Ala Ile Gln Glu Leu Leu SerIle Tyr Asp Cys Arg Glu Met Glu Thr 1420 1425 1430 1435 aac ggc cca ggtcac caa ttg tgg agg aga ttt cct gag cat gtt cgg 4432 Asn Gly Pro Gly HisGln Leu Trp Arg Arg Phe Pro Glu His Val Arg 1440 1445 1450 gaa ata ctagaa cct cat cta aat acc aga tac aag agt tct cag aag 4480 Glu Ile Leu GluPro His Leu Asn Thr Arg Tyr Lys Ser Ser Gln Lys 1455 1460 1465 tca accgat tgg tct gga gta aag aag cca att tac tta agt aaa ttg 4528 Ser Thr AspTrp Ser Gly Val Lys Lys Pro Ile Tyr Leu Ser Lys Leu 1470 1475 1480 ggtagt aac ttt gca gaa tgg tca gca tct tgg gca ggt tat ctt att 4576 Gly SerAsn Phe Ala Glu Trp Ser Ala Ser Trp Ala Gly Tyr Leu Ile 1485 1490 1495aca aag gtt cga cat gat ctt gcc agt aaa att ttc acc tgc tgt agc 4624 ThrLys Val Arg His Asp Leu Ala Ser Lys Ile Phe Thr Cys Cys Ser 1500 15051510 1515 att atg atg aag cat gat ttc aaa gtg acc atc tat ctt ctt ccacat 4672 Ile Met Met Lys His Asp Phe Lys Val Thr Ile Tyr Leu Leu Pro His1520 1525 1530 att ctg gtg tat gtc tta ctg ggt tgt aat caa gaa gat cagcag gag 4720 Ile Leu Val Tyr Val Leu Leu Gly Cys Asn Gln Glu Asp Gln GlnGlu 1535 1540 1545 gtt tat gca gaa att atg gca gtt cta aag cat gac gatcag cat acc 4768 Val Tyr Ala Glu Ile Met Ala Val Leu Lys His Asp Asp GlnHis Thr 1550 1555 1560 ata aat acc caa gac att gca tct gat ctg tgt caactc agt aca cag 4816 Ile Asn Thr Gln Asp Ile Ala Ser Asp Leu Cys Gln LeuSer Thr Gln 1565 1570 1575 act gtg ttc tcc atg ctt gac cat ctc aca cagtgg gca agg cac aaa 4864 Thr Val Phe Ser Met Leu Asp His Leu Thr Gln TrpAla Arg His Lys 1580 1585 1590 1595 ttt cag gca ctg aaa gct gag aaa tgtcca cac agc aaa tca aac aga 4912 Phe Gln Ala Leu Lys Ala Glu Lys Cys ProHis Ser Lys Ser Asn Arg 1600 1605 1610 aat aag gta gac tca atg gta tctact gtg gat tat gaa gac tat cag 4960 Asn Lys Val Asp Ser Met Val Ser ThrVal Asp Tyr Glu Asp Tyr Gln 1615 1620 1625 agt gta acc cgt ttt cta gacctc ata ccc cag gat act ctg gca gta 5008 Ser Val Thr Arg Phe Leu Asp LeuIle Pro Gln Asp Thr Leu Ala Val 1630 1635 1640 gct tcc ttt cgc tcc aaagca tac aca cga gct gta atg cac ttt gaa 5056 Ala Ser Phe Arg Ser Lys AlaTyr Thr Arg Ala Val Met His Phe Glu 1645 1650 1655 tca ttt att aca gaaaag aag caa aat att cag gaa cat ctt gga ttt 5104 Ser Phe Ile Thr Glu LysLys Gln Asn Ile Gln Glu His Leu Gly Phe 1660 1665 1670 1675 tta cag aaattg tat gct gct atg cat gaa cct gat gga gtg gcc gga 5152 Leu Gln Lys LeuTyr Ala Ala Met His Glu Pro Asp Gly Val Ala Gly 1680 1685 1690 gtc agtgca att aga aag gca gaa cca tct cta aaa gaa cag atc ctt 5200 Val Ser AlaIle Arg Lys Ala Glu Pro Ser Leu Lys Glu Gln Ile Leu 1695 1700 1705 gaacat gaa agc ctt ggc ttg ctg agg gat gcc act gct tgt tat gac 5248 Glu HisGlu Ser Leu Gly Leu Leu Arg Asp Ala Thr Ala Cys Tyr Asp 1710 1715 1720agg gct att cag cta gaa cca gac cag atc att cat tat cat ggt gta 5296 ArgAla Ile Gln Leu Glu Pro Asp Gln Ile Ile His Tyr His Gly Val 1725 17301735 gta aag tcc atg tta ggt ctt ggt cag ctg tct act gtt atc act cag5344 Val Lys Ser Met Leu Gly Leu Gly Gln Leu Ser Thr Val Ile Thr Gln1740 1745 1750 1755 gtg aat gga gtg cat gct aac agg tcc gag tgg aca gatgaa tta aac 5392 Val Asn Gly Val His Ala Asn Arg Ser Glu Trp Thr Asp GluLeu Asn 1760 1765 1770 acg tac aga gtg gaa gca gct tgg aaa ttg tca cagtgg gat ttg gtg 5440 Thr Tyr Arg Val Glu Ala Ala Trp Lys Leu Ser Gln TrpAsp Leu Val 1775 1780 1785 gaa aac tat ttg gca gca gat gga aaa tct acaaca tgg agt gtc aga 5488 Glu Asn Tyr Leu Ala Ala Asp Gly Lys Ser Thr ThrTrp Ser Val Arg 1790 1795 1800 ctg gga cag cta tta tta tca gcc aaa aaaaga gat atc aca gct ttt 5536 Leu Gly Gln Leu Leu Leu Ser Ala Lys Lys ArgAsp Ile Thr Ala Phe 1805 1810 1815 tat gac tca ctg aaa cta gtg aga gcagaa caa att gta cct ctt tca 5584 Tyr Asp Ser Leu Lys Leu Val Arg Ala GluGln Ile Val Pro Leu Ser 1820 1825 1830 1835 gct gca agc ttt gaa aga ggctcc tac caa cga gga tat gaa tat att 5632 Ala Ala Ser Phe Glu Arg Gly SerTyr Gln Arg Gly Tyr Glu Tyr Ile 1840 1845 1850 gtg aga ttg cac atg ttatgt gag ttg gag cat agc atc aaa cca ctt 5680 Val Arg Leu His Met Leu CysGlu Leu Glu His Ser Ile Lys Pro Leu 1855 1860 1865 ttc cag cat tct ccaggt gac agt tct caa gaa gat tct cta aac tgg 5728 Phe Gln His Ser Pro GlyAsp Ser Ser Gln Glu Asp Ser Leu Asn Trp 1870 1875 1880 gta gct cga ctagaa atg acc cag aat tcc tac aga gcc aag gac cct 5776 Val Ala Arg Leu GluMet Thr Gln Asn Ser Tyr Arg Ala Lys Asp Pro 1885 1890 1895 atc ctg gctctc cgg agg gct tta cta agc ctc aac aaa aga cca gat 5824 Ile Leu Ala LeuArg Arg Ala Leu Leu Ser Leu Asn Lys Arg Pro Asp 1900 1905 1910 1915 tacaat gaa atg gtt gga gaa tgc tgg ctg cag agt gcc agg gta gct 5872 Tyr AsnGlu Met Val Gly Glu Cys Trp Leu Gln Ser Ala Arg Val Ala 1920 1925 1930aga aag gct ggt cac cac cag aca gcc tac aat gct ctc ctt aat gca 5920 ArgLys Ala Gly His His Gln Thr Ala Tyr Asn Ala Leu Leu Asn Ala 1935 19401945 ggg gaa tca cga ctc gct gaa ctg tac gtg gaa agg gca aag tgg ctc5968 Gly Glu Ser Arg Leu Ala Glu Leu Tyr Val Glu Arg Ala Lys Trp Leu1950 1955 1960 tgg tcc aag ggt gat gtt cac cag gca cta att gtt ctt caaaaa ggt 6016 Trp Ser Lys Gly Asp Val His Gln Ala Leu Ile Val Leu Gln LysGly 1965 1970 1975 gtt gaa tta tgt ttt cct gaa aat gaa acc cca cct gagggt aag aac 6064 Val Glu Leu Cys Phe Pro Glu Asn Glu Thr Pro Pro Glu GlyLys Asn 1980 1985 1990 1995 atg tta atc cat ggt cga gct atg cta cta gtgggc cga ttt atg gaa 6112 Met Leu Ile His Gly Arg Ala Met Leu Leu Val GlyArg Phe Met Glu 2000 2005 2010 gaa aca gct aac ttt gaa agc aat gca attatg aaa aaa tat aag gat 6160 Glu Thr Ala Asn Phe Glu Ser Asn Ala Ile MetLys Lys Tyr Lys Asp 2015 2020 2025 gtg acc gcg tgc ctg cca gaa tgg gaggat ggg cat ttt tac ctt gcc 6208 Val Thr Ala Cys Leu Pro Glu Trp Glu AspGly His Phe Tyr Leu Ala 2030 2035 2040 aag tac tat gac aaa ttg atg cccatg gtc aca gac aac aaa atg gaa 6256 Lys Tyr Tyr Asp Lys Leu Met Pro MetVal Thr Asp Asn Lys Met Glu 2045 2050 2055 aag caa ggt gat ctc atc cggtat ata gtt ctt cat ttt ggc aga tct 6304 Lys Gln Gly Asp Leu Ile Arg TyrIle Val Leu His Phe Gly Arg Ser 2060 2065 2070 2075 cta caa tat gga aatcag ttc ata tat cag tca atg cca cga atg tta 6352 Leu Gln Tyr Gly Asn GlnPhe Ile Tyr Gln Ser Met Pro Arg Met Leu 2080 2085 2090 act cta tgg cttgat tat ggt aca aag gca tat gaa tgg gaa aaa gct 6400 Thr Leu Trp Leu AspTyr Gly Thr Lys Ala Tyr Glu Trp Glu Lys Ala 2095 2100 2105 ggc cgc tccgat cgt gta caa atg agg aat gat ttg ggt aaa ata aac 6448 Gly Arg Ser AspArg Val Gln Met Arg Asn Asp Leu Gly Lys Ile Asn 2110 2115 2120 aag gttatc aca gag cat aca aac tat tta gct cca tat caa ttt ttg 6496 Lys Val IleThr Glu His Thr Asn Tyr Leu Ala Pro Tyr Gln Phe Leu 2125 2130 2135 actgct ttt tca caa ttg atc tct cga att tgt cat tct cac gat gaa 6544 Thr AlaPhe Ser Gln Leu Ile Ser Arg Ile Cys His Ser His Asp Glu 2140 2145 21502155 gtt ttt gtt gtc ttg atg gaa ata ata gcc aaa gta ttt cta gcc tat6592 Val Phe Val Val Leu Met Glu Ile Ile Ala Lys Val Phe Leu Ala Tyr2160 2165 2170 cct caa caa gca atg tgg atg atg aca gct gtg tca aag tcatct tat 6640 Pro Gln Gln Ala Met Trp Met Met Thr Ala Val Ser Lys Ser SerTyr 2175 2180 2185 ccc atg cgt gtg aac aga tgc aag gaa atc ctc aat aaagct att cat 6688 Pro Met Arg Val Asn Arg Cys Lys Glu Ile Leu Asn Lys AlaIle His 2190 2195 2200 atg aaa aaa tcc tta gag aag ttt gtt gga gat gcaact cgc cta aca 6736 Met Lys Lys Ser Leu Glu Lys Phe Val Gly Asp Ala ThrArg Leu Thr 2205 2210 2215 gat aag ctt cta gaa ttg tgc aat aaa ccg gttgat gga agt agt tcc 6784 Asp Lys Leu Leu Glu Leu Cys Asn Lys Pro Val AspGly Ser Ser Ser 2220 2225 2230 2235 aca tta agc atg agc act cat ttt aaaatg ctt aaa aag ctg gta gaa 6832 Thr Leu Ser Met Ser Thr His Phe Lys MetLeu Lys Lys Leu Val Glu 2240 2245 2250 gaa gca aca ttt agt gaa atc ctcatt cct cta caa tca gtc atg ata 6880 Glu Ala Thr Phe Ser Glu Ile Leu IlePro Leu Gln Ser Val Met Ile 2255 2260 2265 cct aca ctt cca tca att ctgggt acc cat gct aac cat gct agc cat 6928 Pro Thr Leu Pro Ser Ile Leu GlyThr His Ala Asn His Ala Ser His 2270 2275 2280 gaa cca ttt cct gga cattgg gcc tat att gca ggg ttt gat gat atg 6976 Glu Pro Phe Pro Gly His TrpAla Tyr Ile Ala Gly Phe Asp Asp Met 2285 2290 2295 gtg gaa att ctt gcttct ctt cag aaa cca aag aag att tct tta aaa 7024 Val Glu Ile Leu Ala SerLeu Gln Lys Pro Lys Lys Ile Ser Leu Lys 2300 2305 2310 2315 ggc tca gatgga aag ttc tac atc atg atg tgt aag cca aaa gat gac 7072 Gly Ser Asp GlyLys Phe Tyr Ile Met Met Cys Lys Pro Lys Asp Asp 2320 2325 2330 ctg agaaag gat tgt aga cta atg gaa ttc aat tcc ttg att aat aag 7120 Leu Arg LysAsp Cys Arg Leu Met Glu Phe Asn Ser Leu Ile Asn Lys 2335 2340 2345 tgctta aga aaa gat gca gag tct cgt aga aga gaa ctt cat att cga 7168 Cys LeuArg Lys Asp Ala Glu Ser Arg Arg Arg Glu Leu His Ile Arg 2350 2355 2360aca tat gca gtt att cca cta aat gat gaa tgt ggg att att gaa tgg 7216 ThrTyr Ala Val Ile Pro Leu Asn Asp Glu Cys Gly Ile Ile Glu Trp 2365 23702375 gtg aac aac act gct ggt ttg aga cct att ctg acc aaa cta tat aaa7264 Val Asn Asn Thr Ala Gly Leu Arg Pro Ile Leu Thr Lys Leu Tyr Lys2380 2385 2390 2395 gaa aag gga gtg tat atg aca gga aaa gaa ctt cgc cagtgt atg cta 7312 Glu Lys Gly Val Tyr Met Thr Gly Lys Glu Leu Arg Gln CysMet Leu 2400 2405 2410 cca aag tca gca gct tta tct gaa aaa ctc aaa gtattc cga gaa ttt 7360 Pro Lys Ser Ala Ala Leu Ser Glu Lys Leu Lys Val PheArg Glu Phe 2415 2420 2425 ctc ctg ccc agg cat cct cct att ttt cat gagtgg ttt ctg aga aca 7408 Leu Leu Pro Arg His Pro Pro Ile Phe His Glu TrpPhe Leu Arg Thr 2430 2435 2440 ttc cct gat cct aca tca tgg tac agt agtaga tca gct tac tgc cgt 7456 Phe Pro Asp Pro Thr Ser Trp Tyr Ser Ser ArgSer Ala Tyr Cys Arg 2445 2450 2455 tcc act gca gta atg tca atg gtt ggttat att ctg ggg ctt gga gac 7504 Ser Thr Ala Val Met Ser Met Val Gly TyrIle Leu Gly Leu Gly Asp 2460 2465 2470 2475 cgt cat ggt gaa aat att ctcttt gat tct ttg act ggt gaa tgc gta 7552 Arg His Gly Glu Asn Ile Leu PheAsp Ser Leu Thr Gly Glu Cys Val 2480 2485 2490 cat gta gat ttc aat tgtctt ttc aat aag gga gaa acc ttt gaa gtt 7600 His Val Asp Phe Asn Cys LeuPhe Asn Lys Gly Glu Thr Phe Glu Val 2495 2500 2505 cca gaa att gtg ccattt cgc ctg act cat aat atg gtt aat gga atg 7648 Pro Glu Ile Val Pro PheArg Leu Thr His Asn Met Val Asn Gly Met 2510 2515 2520 ggt cct atg ggaaca gag ggt ctt ttt cga aga gca tgt gaa gtt aca 7696 Gly Pro Met Gly ThrGlu Gly Leu Phe Arg Arg Ala Cys Glu Val Thr 2525 2530 2535 atg agg ctgatg cgt gat cag cga gag cct tta atg agt gtc tta aag 7744 Met Arg Leu MetArg Asp Gln Arg Glu Pro Leu Met Ser Val Leu Lys 2540 2545 2550 2555 actttt cta cat gat cct ctt gtg gaa tgg agt aaa cca gtg aaa ggg 7792 Thr PheLeu His Asp Pro Leu Val Glu Trp Ser Lys Pro Val Lys Gly 2560 2565 2570cat tcc aaa gcg cca ctg aat gaa act gga gaa gtt gtc aat gaa aag 7840 HisSer Lys Ala Pro Leu Asn Glu Thr Gly Glu Val Val Asn Glu Lys 2575 25802585 gcc aag acc cat gtt ctt gac att gag cag cga cta caa ggt gta atc7888 Ala Lys Thr His Val Leu Asp Ile Glu Gln Arg Leu Gln Gly Val Ile2590 2595 2600 aag act cga aat aga gtg aca gga ctg ccg tta tct att gaagga cat 7936 Lys Thr Arg Asn Arg Val Thr Gly Leu Pro Leu Ser Ile Glu GlyHis 2605 2610 2615 gtg cat tac ctt ata caa gaa gct act gat gaa aac ttacta tgc cag 7984 Val His Tyr Leu Ile Gln Glu Ala Thr Asp Glu Asn Leu LeuCys Gln 2620 2625 2630 2635 atg tat ctt ggt tgg act cca tat atgtgaaatgaaa ttatgtaaaa 8031 Met Tyr Leu Gly Trp Thr Pro Tyr Met 2640gaatatgtta ataatctaaa agtaatgcat ttggtatgaa tctgtggttg tatctgttca 8091attctaaagt acaacataaa tttacgttct cagcaactgt tatttctctc tgatcattaa 8151ttatatgtaa aataatatac attcagttat taagaaataa actgctttct taataaaaaa 8211aaaaaaaaaa aaaaaaaaaa aaaaaaaa 8239 2 2644 PRT Homo sapiens 2 Met GlyGlu His Gly Leu Glu Leu Ala Ser Met Ile Pro Ala Leu Arg 1 5 10 15 GluLeu Gly Ser Ala Thr Pro Glu Glu Tyr Asn Thr Val Val Gln Lys 20 25 30 ProArg Gln Ile Leu Cys Gln Phe Ile Asp Arg Ile Leu Thr Asp Val 35 40 45 AsnVal Val Ala Val Glu Leu Val Lys Lys Thr Asp Ser Gln Pro Thr 50 55 60 SerVal Met Leu Leu Asp Phe Ile Gln His Ile Met Lys Ser Ser Pro 65 70 75 80Leu Met Phe Val Asn Val Ser Gly Ser His Glu Arg Lys Gly Ser Cys 85 90 95Ile Glu Phe Ser Asn Trp Ile Ile Thr Arg Leu Leu Arg Ile Ala Ala 100 105110 Thr Pro Ser Cys His Leu Leu His Lys Lys Ile Cys Glu Val Ile Cys 115120 125 Ser Leu Leu Phe Leu Phe Lys Ser Lys Ser Pro Ala Ile Phe Gly Val130 135 140 Leu Thr Lys Glu Leu Leu Gln Leu Phe Glu Asp Leu Val Tyr LeuHis 145 150 155 160 Arg Arg Asn Val Met Gly His Ala Val Glu Trp Pro ValVal Met Ser 165 170 175 Arg Phe Leu Ser Gln Leu Asp Glu His Met Gly TyrLeu Gln Ser Ala 180 185 190 Pro Leu Gln Leu Met Ser Met Gln Asn Leu GluPhe Ile Glu Val Thr 195 200 205 Leu Leu Met Val Leu Thr Arg Ile Ile AlaIle Val Phe Phe Arg Arg 210 215 220 Gln Glu Leu Leu Leu Trp Gln Ile GlyCys Val Leu Leu Glu Tyr Gly 225 230 235 240 Ser Pro Lys Ile Lys Ser LeuAla Ile Ser Phe Leu Thr Glu Leu Phe 245 250 255 Gln Leu Gly Gly Leu ProAla Gln Pro Ala Ser Thr Phe Phe Ser Ser 260 265 270 Phe Leu Glu Leu LeuLys His Leu Val Glu Met Asp Thr Asp Gln Leu 275 280 285 Lys Leu Tyr GluGlu Pro Leu Ser Lys Leu Ile Lys Thr Leu Phe Pro 290 295 300 Phe Glu AlaGlu Ala Tyr Arg Asn Ile Glu Pro Val Tyr Leu Asn Met 305 310 315 320 LeuLeu Glu Lys Leu Cys Val Met Phe Glu Asp Gly Val Leu Met Arg 325 330 335Leu Lys Ser Asp Leu Leu Lys Ala Ala Leu Cys His Leu Leu Gln Tyr 340 345350 Phe Leu Lys Phe Val Pro Ala Gly Tyr Glu Ser Ala Leu Gln Val Arg 355360 365 Lys Val Tyr Val Arg Asn Ile Cys Lys Ala Leu Leu Asp Val Leu Gly370 375 380 Ile Glu Val Asp Ala Glu Tyr Leu Leu Gly Pro Leu Tyr Ala AlaLeu 385 390 395 400 Lys Met Glu Ser Met Glu Ile Ile Glu Glu Ile Gln CysGln Thr Gln 405 410 415 Gln Glu Asn Leu Ser Ser Asn Ser Asp Gly Ile SerPro Lys Arg Arg 420 425 430 Arg Leu Ser Ser Ser Leu Asn Pro Ser Lys ArgAla Pro Lys Gln Thr 435 440 445 Glu Glu Ile Lys His Val Asp Met Asn GlnLys Ser Ile Leu Trp Ser 450 455 460 Ala Leu Lys Gln Lys Ala Glu Ser LeuGln Ile Ser Leu Glu Tyr Ser 465 470 475 480 Gly Leu Lys Asn Pro Val IleGlu Met Leu Glu Gly Ile Ala Val Val 485 490 495 Leu Gln Leu Thr Ala LeuCys Thr Val His Cys Ser His Gln Asn Met 500 505 510 Asn Cys Arg Thr PheLys Asp Cys Gln His Lys Ser Lys Lys Lys Pro 515 520 525 Ser Val Val IleThr Trp Met Ser Leu Asp Phe Tyr Thr Lys Val Leu 530 535 540 Lys Ser CysArg Ser Leu Leu Glu Ser Val Gln Lys Leu Asp Leu Glu 545 550 555 560 AlaThr Ile Asp Lys Val Val Lys Ile Tyr Asp Ala Leu Ile Tyr Met 565 570 575Gln Val Asn Ser Ser Phe Glu Asp His Ile Leu Glu Asp Leu Cys Gly 580 585590 Met Leu Ser Leu Pro Trp Ile Tyr Ser His Ser Asp Asp Gly Cys Leu 595600 605 Lys Leu Thr Thr Phe Ala Ala Asn Leu Leu Thr Leu Ser Cys Arg Ile610 615 620 Ser Asp Ser Tyr Ser Pro Gln Ala Gln Ser Arg Cys Val Phe LeuLeu 625 630 635 640 Thr Leu Phe Pro Arg Arg Ile Phe Leu Glu Trp Arg ThrAla Val Tyr 645 650 655 Asn Trp Ala Leu Gln Ser Ser His Glu Val Ile ArgAla Ser Cys Val 660 665 670 Ser Gly Phe Phe Ile Leu Leu Gln Gln Gln AsnSer Cys Asn Arg Val 675 680 685 Pro Lys Ile Leu Ile Asp Lys Val Lys AspAsp Ser Asp Ile Val Lys 690 695 700 Lys Glu Phe Ala Ser Ile Leu Gly GlnLeu Val Cys Thr Leu His Gly 705 710 715 720 Met Phe Tyr Leu Thr Ser SerLeu Thr Glu Pro Phe Ser Glu His Gly 725 730 735 His Val Asp Leu Phe CysArg Asn Leu Lys Ala Thr Ser Gln His Glu 740 745 750 Cys Ser Ser Ser GlnLeu Lys Ala Ser Val Cys Lys Pro Phe Leu Phe 755 760 765 Leu Leu Lys LysLys Ile Pro Ser Pro Val Lys Leu Ala Phe Ile Asp 770 775 780 Asn Leu HisHis Leu Cys Lys His Leu Asp Phe Arg Glu Asp Glu Thr 785 790 795 800 AspVal Lys Ala Val Leu Gly Thr Leu Leu Asn Leu Met Glu Asp Pro 805 810 815Asp Lys Asp Val Arg Val Ala Phe Ser Gly Asn Ile Lys His Ile Leu 820 825830 Glu Ser Leu Asp Ser Glu Asp Gly Phe Ile Lys Glu Leu Phe Val Leu 835840 845 Arg Met Lys Glu Ala Tyr Thr His Ala Gln Ile Ser Arg Asn Asn Glu850 855 860 Leu Lys Asp Thr Leu Ile Leu Thr Thr Gly Asp Ile Gly Arg AlaAla 865 870 875 880 Lys Gly Asp Leu Val Pro Phe Ala Leu Leu His Leu LeuHis Cys Leu 885 890 895 Leu Ser Lys Ser Ala Ser Val Ser Gly Ala Ala TyrThr Glu Ile Arg 900 905 910 Ala Leu Val Ala Ala Lys Ser Val Lys Leu GlnSer Phe Phe Ser Gln 915 920 925 Tyr Lys Lys Pro Ile Cys Gln Phe Leu ValGlu Ser Leu His Ser Ser 930 935 940 Gln Met Thr Ala Leu Pro Asn Thr ProCys Gln Asn Ala Asp Val Arg 945 950 955 960 Lys Gln Asp Val Ala His GlnArg Glu Met Ala Leu Asn Thr Leu Ser 965 970 975 Glu Ile Ala Asn Val PheAsp Phe Pro Asp Leu Asn Arg Phe Leu Thr 980 985 990 Arg Thr Leu Gln ValLeu Leu Pro Asp Leu Ala Ala Lys Ala Ser Pro 995 1000 1005 Ala Ala SerAla Leu Ile Arg Thr Leu Gly Lys Gln Leu Asn Val 1010 1015 1020 Asn ArgArg Glu Ile Leu Ile Asn Asn Phe Lys Tyr Ile Phe Ser 1025 1030 1035 HisLeu Val Cys Ser Cys Ser Lys Asp Glu Leu Glu Arg Ala Leu 1040 1045 1050His Tyr Leu Lys Asn Glu Thr Glu Ile Glu Leu Gly Ser Leu Leu 1055 10601065 Arg Gln Asp Phe Gln Gly Leu His Asn Glu Leu Leu Leu Arg Ile 10701075 1080 Gly Glu His Tyr Gln Gln Val Phe Asn Gly Leu Ser Ile Leu Ala1085 1090 1095 Ser Phe Ala Ser Ser Asp Asp Pro Tyr Gln Gly Pro Arg AspIle 1100 1105 1110 Ile Ser Pro Glu Leu Met Ala Asp Tyr Leu Gln Pro LysLeu Leu 1115 1120 1125 Gly Ile Leu Ala Phe Phe Asn Met Gln Leu Leu SerSer Ser Val 1130 1135 1140 Gly Ile Glu Asp Lys Lys Met Ala Leu Asn SerLeu Met Ser Leu 1145 1150 1155 Met Lys Leu Met Gly Pro Lys His Val SerSer Val Arg Val Lys 1160 1165 1170 Met Met Thr Thr Leu Arg Thr Gly LeuArg Phe Lys Asp Asp Phe 1175 1180 1185 Pro Glu Leu Cys Cys Arg Ala TrpAsp Cys Phe Val Arg Cys Leu 1190 1195 1200 Asp His Ala Cys Leu Gly SerLeu Leu Ser His Val Ile Val Ala 1205 1210 1215 Leu Leu Pro Leu Ile HisIle Gln Pro Lys Glu Thr Ala Ala Ile 1220 1225 1230 Phe His Tyr Leu IleIle Glu Asn Arg Asp Ala Val Gln Asp Phe 1235 1240 1245 Leu His Glu IleTyr Phe Leu Pro Asp His Pro Glu Leu Lys Lys 1250 1255 1260 Ile Lys AlaVal Leu Gln Glu Tyr Arg Lys Glu Thr Ser Glu Ser 1265 1270 1275 Thr AspLeu Gln Thr Thr Leu Gln Leu Ser Met Lys Ala Ile Gln 1280 1285 1290 HisGlu Asn Val Asp Val Arg Ile His Ala Leu Thr Ser Leu Lys 1295 1300 1305Glu Thr Leu Tyr Lys Asn Gln Glu Lys Leu Ile Lys Tyr Ala Thr 1310 13151320 Asp Ser Glu Thr Val Glu Pro Ile Ile Ser Gln Leu Val Thr Val 13251330 1335 Leu Leu Lys Gly Cys Gln Asp Ala Asn Ser Gln Ala Arg Leu Leu1340 1345 1350 Cys Gly Glu Cys Leu Gly Glu Leu Gly Ala Ile Asp Pro GlyArg 1355 1360 1365 Leu Asp Phe Ser Thr Thr Glu Thr Gln Gly Lys Asp PheThr Phe 1370 1375 1380 Val Thr Gly Val Glu Asp Ser Ser Phe Ala Tyr GlyLeu Leu Met 1385 1390 1395 Glu Leu Thr Arg Ala Tyr Leu Ala Tyr Ala AspAsn Ser Arg Ala 1400 1405 1410 Gln Asp Ser Ala Ala Tyr Ala Ile Gln GluLeu Leu Ser Ile Tyr 1415 1420 1425 Asp Cys Arg Glu Met Glu Thr Asn GlyPro Gly His Gln Leu Trp 1430 1435 1440 Arg Arg Phe Pro Glu His Val ArgGlu Ile Leu Glu Pro His Leu 1445 1450 1455 Asn Thr Arg Tyr Lys Ser SerGln Lys Ser Thr Asp Trp Ser Gly 1460 1465 1470 Val Lys Lys Pro Ile TyrLeu Ser Lys Leu Gly Ser Asn Phe Ala 1475 1480 1485 Glu Trp Ser Ala SerTrp Ala Gly Tyr Leu Ile Thr Lys Val Arg 1490 1495 1500 His Asp Leu AlaSer Lys Ile Phe Thr Cys Cys Ser Ile Met Met 1505 1510 1515 Lys His AspPhe Lys Val Thr Ile Tyr Leu Leu Pro His Ile Leu 1520 1525 1530 Val TyrVal Leu Leu Gly Cys Asn Gln Glu Asp Gln Gln Glu Val 1535 1540 1545 TyrAla Glu Ile Met Ala Val Leu Lys His Asp Asp Gln His Thr 1550 1555 1560Ile Asn Thr Gln Asp Ile Ala Ser Asp Leu Cys Gln Leu Ser Thr 1565 15701575 Gln Thr Val Phe Ser Met Leu Asp His Leu Thr Gln Trp Ala Arg 15801585 1590 His Lys Phe Gln Ala Leu Lys Ala Glu Lys Cys Pro His Ser Lys1595 1600 1605 Ser Asn Arg Asn Lys Val Asp Ser Met Val Ser Thr Val AspTyr 1610 1615 1620 Glu Asp Tyr Gln Ser Val Thr Arg Phe Leu Asp Leu IlePro Gln 1625 1630 1635 Asp Thr Leu Ala Val Ala Ser Phe Arg Ser Lys AlaTyr Thr Arg 1640 1645 1650 Ala Val Met His Phe Glu Ser Phe Ile Thr GluLys Lys Gln Asn 1655 1660 1665 Ile Gln Glu His Leu Gly Phe Leu Gln LysLeu Tyr Ala Ala Met 1670 1675 1680 His Glu Pro Asp Gly Val Ala Gly ValSer Ala Ile Arg Lys Ala 1685 1690 1695 Glu Pro Ser Leu Lys Glu Gln IleLeu Glu His Glu Ser Leu Gly 1700 1705 1710 Leu Leu Arg Asp Ala Thr AlaCys Tyr Asp Arg Ala Ile Gln Leu 1715 1720 1725 Glu Pro Asp Gln Ile IleHis Tyr His Gly Val Val Lys Ser Met 1730 1735 1740 Leu Gly Leu Gly GlnLeu Ser Thr Val Ile Thr Gln Val Asn Gly 1745 1750 1755 Val His Ala AsnArg Ser Glu Trp Thr Asp Glu Leu Asn Thr Tyr 1760 1765 1770 Arg Val GluAla Ala Trp Lys Leu Ser Gln Trp Asp Leu Val Glu 1775 1780 1785 Asn TyrLeu Ala Ala Asp Gly Lys Ser Thr Thr Trp Ser Val Arg 1790 1795 1800 LeuGly Gln Leu Leu Leu Ser Ala Lys Lys Arg Asp Ile Thr Ala 1805 1810 1815Phe Tyr Asp Ser Leu Lys Leu Val Arg Ala Glu Gln Ile Val Pro 1820 18251830 Leu Ser Ala Ala Ser Phe Glu Arg Gly Ser Tyr Gln Arg Gly Tyr 18351840 1845 Glu Tyr Ile Val Arg Leu His Met Leu Cys Glu Leu Glu His Ser1850 1855 1860 Ile Lys Pro Leu Phe Gln His Ser Pro Gly Asp Ser Ser GlnGlu 1865 1870 1875 Asp Ser Leu Asn Trp Val Ala Arg Leu Glu Met Thr GlnAsn Ser 1880 1885 1890 Tyr Arg Ala Lys Asp Pro Ile Leu Ala Leu Arg ArgAla Leu Leu 1895 1900 1905 Ser Leu Asn Lys Arg Pro Asp Tyr Asn Glu MetVal Gly Glu Cys 1910 1915 1920 Trp Leu Gln Ser Ala Arg Val Ala Arg LysAla Gly His His Gln 1925 1930 1935 Thr Ala Tyr Asn Ala Leu Leu Asn AlaGly Glu Ser Arg Leu Ala 1940 1945 1950 Glu Leu Tyr Val Glu Arg Ala LysTrp Leu Trp Ser Lys Gly Asp 1955 1960 1965 Val His Gln Ala Leu Ile ValLeu Gln Lys Gly Val Glu Leu Cys 1970 1975 1980 Phe Pro Glu Asn Glu ThrPro Pro Glu Gly Lys Asn Met Leu Ile 1985 1990 1995 His Gly Arg Ala MetLeu Leu Val Gly Arg Phe Met Glu Glu Thr 2000 2005 2010 Ala Asn Phe GluSer Asn Ala Ile Met Lys Lys Tyr Lys Asp Val 2015 2020 2025 Thr Ala CysLeu Pro Glu Trp Glu Asp Gly His Phe Tyr Leu Ala 2030 2035 2040 Lys TyrTyr Asp Lys Leu Met Pro Met Val Thr Asp Asn Lys Met 2045 2050 2055 GluLys Gln Gly Asp Leu Ile Arg Tyr Ile Val Leu His Phe Gly 2060 2065 2070Arg Ser Leu Gln Tyr Gly Asn Gln Phe Ile Tyr Gln Ser Met Pro 2075 20802085 Arg Met Leu Thr Leu Trp Leu Asp Tyr Gly Thr Lys Ala Tyr Glu 20902095 2100 Trp Glu Lys Ala Gly Arg Ser Asp Arg Val Gln Met Arg Asn Asp2105 2110 2115 Leu Gly Lys Ile Asn Lys Val Ile Thr Glu His Thr Asn TyrLeu 2120 2125 2130 Ala Pro Tyr Gln Phe Leu Thr Ala Phe Ser Gln Leu IleSer Arg 2135 2140 2145 Ile Cys His Ser His Asp Glu Val Phe Val Val LeuMet Glu Ile 2150 2155 2160 Ile Ala Lys Val Phe Leu Ala Tyr Pro Gln GlnAla Met Trp Met 2165 2170 2175 Met Thr Ala Val Ser Lys Ser Ser Tyr ProMet Arg Val Asn Arg 2180 2185 2190 Cys Lys Glu Ile Leu Asn Lys Ala IleHis Met Lys Lys Ser Leu 2195 2200 2205 Glu Lys Phe Val Gly Asp Ala ThrArg Leu Thr Asp Lys Leu Leu 2210 2215 2220 Glu Leu Cys Asn Lys Pro ValAsp Gly Ser Ser Ser Thr Leu Ser 2225 2230 2235 Met Ser Thr His Phe LysMet Leu Lys Lys Leu Val Glu Glu Ala 2240 2245 2250 Thr Phe Ser Glu IleLeu Ile Pro Leu Gln Ser Val Met Ile Pro 2255 2260 2265 Thr Leu Pro SerIle Leu Gly Thr His Ala Asn His Ala Ser His 2270 2275 2280 Glu Pro PhePro Gly His Trp Ala Tyr Ile Ala Gly Phe Asp Asp 2285 2290 2295 Met ValGlu Ile Leu Ala Ser Leu Gln Lys Pro Lys Lys Ile Ser 2300 2305 2310 LeuLys Gly Ser Asp Gly Lys Phe Tyr Ile Met Met Cys Lys Pro 2315 2320 2325Lys Asp Asp Leu Arg Lys Asp Cys Arg Leu Met Glu Phe Asn Ser 2330 23352340 Leu Ile Asn Lys Cys Leu Arg Lys Asp Ala Glu Ser Arg Arg Arg 23452350 2355 Glu Leu His Ile Arg Thr Tyr Ala Val Ile Pro Leu Asn Asp Glu2360 2365 2370 Cys Gly Ile Ile Glu Trp Val Asn Asn Thr Ala Gly Leu ArgPro 2375 2380 2385 Ile Leu Thr Lys Leu Tyr Lys Glu Lys Gly Val Tyr MetThr Gly 2390 2395 2400 Lys Glu Leu Arg Gln Cys Met Leu Pro Lys Ser AlaAla Leu Ser 2405 2410 2415 Glu Lys Leu Lys Val Phe Arg Glu Phe Leu LeuPro Arg His Pro 2420 2425 2430 Pro Ile Phe His Glu Trp Phe Leu Arg ThrPhe Pro Asp Pro Thr 2435 2440 2445 Ser Trp Tyr Ser Ser Arg Ser Ala TyrCys Arg Ser Thr Ala Val 2450 2455 2460 Met Ser Met Val Gly Tyr Ile LeuGly Leu Gly Asp Arg His Gly 2465 2470 2475 Glu Asn Ile Leu Phe Asp SerLeu Thr Gly Glu Cys Val His Val 2480 2485 2490 Asp Phe Asn Cys Leu PheAsn Lys Gly Glu Thr Phe Glu Val Pro 2495 2500 2505 Glu Ile Val Pro PheArg Leu Thr His Asn Met Val Asn Gly Met 2510 2515 2520 Gly Pro Met GlyThr Glu Gly Leu Phe Arg Arg Ala Cys Glu Val 2525 2530 2535 Thr Met ArgLeu Met Arg Asp Gln Arg Glu Pro Leu Met Ser Val 2540 2545 2550 Leu LysThr Phe Leu His Asp Pro Leu Val Glu Trp Ser Lys Pro 2555 2560 2565 ValLys Gly His Ser Lys Ala Pro Leu Asn Glu Thr Gly Glu Val 2570 2575 2580Val Asn Glu Lys Ala Lys Thr His Val Leu Asp Ile Glu Gln Arg 2585 25902595 Leu Gln Gly Val Ile Lys Thr Arg Asn Arg Val Thr Gly Leu Pro 26002605 2610 Leu Ser Ile Glu Gly His Val His Tyr Leu Ile Gln Glu Ala Thr2615 2620 2625 Asp Glu Asn Leu Leu Cys Gln Met Tyr Leu Gly Trp Thr ProTyr 2630 2635 2640 Met 3 8022 DNA Homo sapiens CDS (585)..(7742) 3ggtaccaagt aaaaactgct tagtaagtat aaaacacaga agaatccgcg atctagtgaa 60ccaatgccct gcgtatgacg ctccactgac gctatagtca atgagaacta ggatgtgcga 120ttataactta tcttttcaat attttcttat tatttattta agaaataatt gaattaaaac 180tcatttcttc ttttattagc cgtaaaatag cttattttct ctcctactac ctttcaacaa 240taactttttt ttttgtttat tgaccattat aatcacatca aaagtcaaaa aattcaatca 300ttatcagaaa catccagcct aatattactt aaaagttagt ttcctctgaa aattcagtat 360cacaaaagct cgttaattag catcgctcga tacttagtgc accatgcatc ttcctttacc 420tcgtgagtgg aaatcgattt gataatcgat tgccactttt cgcataattc tattgagata 480ttttattact tacaatcgtc ttttataaat gctcaagact ttgaacgcgc gtgttgcgtt 540ttaaaaaggc ctttttttga attgaatcaa tggtttgata tagt atg agc caa cac 596 MetSer Gln His 1 gca aaa agg aaa gct ggg tca ctc gat ctt tca ccc aga ggctta gat 644 Ala Lys Arg Lys Ala Gly Ser Leu Asp Leu Ser Pro Arg Gly LeuAsp 5 10 15 20 gac aga cag gct ttc gga cag ctt ttg aaa gaa gta tta gcatta gac 692 Asp Arg Gln Ala Phe Gly Gln Leu Leu Lys Glu Val Leu Ala LeuAsp 25 30 35 aaa gaa cat gag tta ggt aga agt aat tct tta cca tct atg acctcc 740 Lys Glu His Glu Leu Gly Arg Ser Asn Ser Leu Pro Ser Met Thr Ser40 45 50 gag ctt gtt gaa gtt tta att gaa gtt ggt ctt cta gct ttt aaa cat788 Glu Leu Val Glu Val Leu Ile Glu Val Gly Leu Leu Ala Phe Lys His 5560 65 gat gat tca aaa tct gaa ttt atc tct cct aag atg cta aaa gaa gcc836 Asp Asp Ser Lys Ser Glu Phe Ile Ser Pro Lys Met Leu Lys Glu Ala 7075 80 cat ctc tct cta caa gcg tta atg cta atc tta aaa agg tct ccg aca884 His Leu Ser Leu Gln Ala Leu Met Leu Ile Leu Lys Arg Ser Pro Thr 8590 95 100 gtt ttg cgg gag att aaa tca tct gtt act ctt ttg gat tgg atttta 932 Val Leu Arg Glu Ile Lys Ser Ser Val Thr Leu Leu Asp Trp Ile Leu105 110 115 ccc agg act ata tca ttg ttt gct gat att cgt ttt att aag ttattt 980 Pro Arg Thr Ile Ser Leu Phe Ala Asp Ile Arg Phe Ile Lys Leu Phe120 125 130 gac tca tta aaa gag ttt cat aag cta att tat cag cta atc agtgaa 1028 Asp Ser Leu Lys Glu Phe His Lys Leu Ile Tyr Gln Leu Ile Ser Glu135 140 145 aag tca ttc cta tgg gac tta tat gct tcg ttt atg cgt tat tggaaa 1076 Lys Ser Phe Leu Trp Asp Leu Tyr Ala Ser Phe Met Arg Tyr Trp Lys150 155 160 tat tat att aca aac gtt tct tct ata gtt ctc caa atc act aatgct 1124 Tyr Tyr Ile Thr Asn Val Ser Ser Ile Val Leu Gln Ile Thr Asn Ala165 170 175 180 aca ttc cct tac aag atg ccc tca ccc aat tct caa cca ttgcag agt 1172 Thr Phe Pro Tyr Lys Met Pro Ser Pro Asn Ser Gln Pro Leu GlnSer 185 190 195 atc tcc cca aat tat cca acc cat cga gag gac aaa ttt gattta ctt 1220 Ile Ser Pro Asn Tyr Pro Thr His Arg Glu Asp Lys Phe Asp LeuLeu 200 205 210 atc att aat ata gag gag gct tgt aca ttt ttc ttt gaa agtgcc cat 1268 Ile Ile Asn Ile Glu Glu Ala Cys Thr Phe Phe Phe Glu Ser AlaHis 215 220 225 ttt ttt gca caa tgc tca tat tta aag aaa tcc aat ttt cctagt cca 1316 Phe Phe Ala Gln Cys Ser Tyr Leu Lys Lys Ser Asn Phe Pro SerPro 230 235 240 cct ctc ttt aca gcg tgg act tgg atc aag cca tgt ttt tttaat ttt 1364 Pro Leu Phe Thr Ala Trp Thr Trp Ile Lys Pro Cys Phe Phe AsnPhe 245 250 255 260 gtt att tta tta aaa cga atc agc atc gga gac tca cagctc ttt cta 1412 Val Ile Leu Leu Lys Arg Ile Ser Ile Gly Asp Ser Gln LeuPhe Leu 265 270 275 cat ttg cat tca cgt ata gtc caa act tta tgc tgt ttttcc ttg aat 1460 His Leu His Ser Arg Ile Val Gln Thr Leu Cys Cys Phe SerLeu Asn 280 285 290 ttt ata tat cat ggc ctt ccc att tgt gaa aaa tct aaacat att tta 1508 Phe Ile Tyr His Gly Leu Pro Ile Cys Glu Lys Ser Lys HisIle Leu 295 300 305 atg tcc tcc atc aac tta aca ttg gga tca ttg aag aaaact tat aca 1556 Met Ser Ser Ile Asn Leu Thr Leu Gly Ser Leu Lys Lys ThrTyr Thr 310 315 320 gtt gct aat act gct ata tct ctt ttt ttt ctc tct ttattt gtt tta 1604 Val Ala Asn Thr Ala Ile Ser Leu Phe Phe Leu Ser Leu PheVal Leu 325 330 335 340 ccc aaa act gta gct ggt cta ttc tat cct ttt ggggtt tcc tta ctt 1652 Pro Lys Thr Val Ala Gly Leu Phe Tyr Pro Phe Gly ValSer Leu Leu 345 350 355 tct gac ttc aag gta tta gag caa ctt gaa cca gattct gat ctc aaa 1700 Ser Asp Phe Lys Val Leu Glu Gln Leu Glu Pro Asp SerAsp Leu Lys 360 365 370 aag gca ata ata tta ttt aag tgc aga tac caa agttca gaa ata gat 1748 Lys Ala Ile Ile Leu Phe Lys Cys Arg Tyr Gln Ser SerGlu Ile Asp 375 380 385 caa aca act ctc cgt gct ttt ggc gaa att tgt actggt aaa ctt gaa 1796 Gln Thr Thr Leu Arg Ala Phe Gly Glu Ile Cys Thr GlyLys Leu Glu 390 395 400 aac acg ttg ttt tct aac tct gaa tta aac ctt tttctt tta cat tat 1844 Asn Thr Leu Phe Ser Asn Ser Glu Leu Asn Leu Phe LeuLeu His Tyr 405 410 415 420 ctt tcc ttg gac aat gac ttg tca aat att cttaaa gtg gat ttc cag 1892 Leu Ser Leu Asp Asn Asp Leu Ser Asn Ile Leu LysVal Asp Phe Gln 425 430 435 aat ggt cat aac ata tgt aca ttt gca aaa tggtgt ata aac aac aac 1940 Asn Gly His Asn Ile Cys Thr Phe Ala Lys Trp CysIle Asn Asn Asn 440 445 450 tta gat gaa ccg tct aat tta aag cac ttt cgtgaa atg tta gat tat 1988 Leu Asp Glu Pro Ser Asn Leu Lys His Phe Arg GluMet Leu Asp Tyr 455 460 465 tat agc tct cat aat gtt aca ata agt gag gacgac ctg aag aac ttc 2036 Tyr Ser Ser His Asn Val Thr Ile Ser Glu Asp AspLeu Lys Asn Phe 470 475 480 tct tta gtt ttg tgt act cat gtt gca aag gtgaat gag aaa aca aat 2084 Ser Leu Val Leu Cys Thr His Val Ala Lys Val AsnGlu Lys Thr Asn 485 490 495 500 agt att ttc cgc aca tat gaa gta cat ggttgt gaa gtt tgt aac tca 2132 Ser Ile Phe Arg Thr Tyr Glu Val His Gly CysGlu Val Cys Asn Ser 505 510 515 ttt tgt tta cta ttt gat gag cgg tcg cctttt aaa att cct tat cac 2180 Phe Cys Leu Leu Phe Asp Glu Arg Ser Pro PheLys Ile Pro Tyr His 520 525 530 gaa ttg ttt tgt gca ttg cta aaa aat cccgac ata att tcc tct tct 2228 Glu Leu Phe Cys Ala Leu Leu Lys Asn Pro AspIle Ile Ser Ser Ser 535 540 545 gtt aaa caa tca ttg ttg ctt gat ggc tttttt cgg tgg agc cag cat 2276 Val Lys Gln Ser Leu Leu Leu Asp Gly Phe PheArg Trp Ser Gln His 550 555 560 tgc tca aac ttt aat aaa gaa tca atg ttaagt tta aga gaa ttt att 2324 Cys Ser Asn Phe Asn Lys Glu Ser Met Leu SerLeu Arg Glu Phe Ile 565 570 575 580 atg aaa gca tta gcc agt act tca agatgt tta cgt gtt gtt gct gca 2372 Met Lys Ala Leu Ala Ser Thr Ser Arg CysLeu Arg Val Val Ala Ala 585 590 595 aaa gtt ttg ccc att ttc att aag ggacct aat aat ctt gat ata gtt 2420 Lys Val Leu Pro Ile Phe Ile Lys Gly ProAsn Asn Leu Asp Ile Val 600 605 610 gaa ttt cac aag gaa agt aaa gcc ttgatt ttt aat acg ttg aaa ata 2468 Glu Phe His Lys Glu Ser Lys Ala Leu IlePhe Asn Thr Leu Lys Ile 615 620 625 ttg gcg gtg gaa aat aca gct att ttagaa acg gtc att ctt tcc tgg 2516 Leu Ala Val Glu Asn Thr Ala Ile Leu GluThr Val Ile Leu Ser Trp 630 635 640 atc tcc tta tct aga gtg gta gaa gaagaa gaa tta cat ttt gta cta 2564 Ile Ser Leu Ser Arg Val Val Glu Glu GluGlu Leu His Phe Val Leu 645 650 655 660 ttg gaa gtt ata tct tct gtg ataaac agc gga ata ttt tat caa ggc 2612 Leu Glu Val Ile Ser Ser Val Ile AsnSer Gly Ile Phe Tyr Gln Gly 665 670 675 att ggt ctc agc gct ctg caa caaatt gcc tcg acg cgt cat ata tcc 2660 Ile Gly Leu Ser Ala Leu Gln Gln IleAla Ser Thr Arg His Ile Ser 680 685 690 gtt tgg caa tta ctt tct cca tattgg cca aca gtg tcc gtt gcg att 2708 Val Trp Gln Leu Leu Ser Pro Tyr TrpPro Thr Val Ser Val Ala Ile 695 700 705 gtc caa ggt atg ggt aaa aaa ccgaac ata gcc agt tta ttt gct cag 2756 Val Gln Gly Met Gly Lys Lys Pro AsnIle Ala Ser Leu Phe Ala Gln 710 715 720 ctt atg aat att tcc gag ggc gatttt ctt att cga aca cag gcg tac 2804 Leu Met Asn Ile Ser Glu Gly Asp PheLeu Ile Arg Thr Gln Ala Tyr 725 730 735 740 act tta cca ttc ctt gta cttact aaa aac aaa gcg tta ata gta cgt 2852 Thr Leu Pro Phe Leu Val Leu ThrLys Asn Lys Ala Leu Ile Val Arg 745 750 755 ata gct gaa ctt tca caa agtgat gtt gct act ttg tgc ctt acc aat 2900 Ile Ala Glu Leu Ser Gln Ser AspVal Ala Thr Leu Cys Leu Thr Asn 760 765 770 atg cat aaa atc ctt gct tcgcta ctt act acg gat cat cct aat ttg 2948 Met His Lys Ile Leu Ala Ser LeuLeu Thr Thr Asp His Pro Asn Leu 775 780 785 gaa gag agt gtg atg ctt cttctt tca ctg gcc act tct gat ttt gaa 2996 Glu Glu Ser Val Met Leu Leu LeuSer Leu Ala Thr Ser Asp Phe Glu 790 795 800 aaa gtt gat tta acg tct ttgtta cgc tct gat cct att tct att act 3044 Lys Val Asp Leu Thr Ser Leu LeuArg Ser Asp Pro Ile Ser Ile Thr 805 810 815 820 gtg gag ttg tta cag ctttat cag aat gat gtt cct cat gaa aaa att 3092 Val Glu Leu Leu Gln Leu TyrGln Asn Asp Val Pro His Glu Lys Ile 825 830 835 gaa aat gct tta aga aaggta gca atg att gtc tct caa gtg gtt aat 3140 Glu Asn Ala Leu Arg Lys ValAla Met Ile Val Ser Gln Val Val Asn 840 845 850 gac gaa gac ttg agc aataag gaa tta ctt tat gat ttt ttt aat aat 3188 Asp Glu Asp Leu Ser Asn LysGlu Leu Leu Tyr Asp Phe Phe Asn Asn 855 860 865 cac att ttg ggt atc ttagca gaa ttt tct aat atc ctt aac gac ctg 3236 His Ile Leu Gly Ile Leu AlaGlu Phe Ser Asn Ile Leu Asn Asp Leu 870 875 880 aaa gga aag act tca attaat gaa aag att aag aca att gtc ggc att 3284 Lys Gly Lys Thr Ser Ile AsnGlu Lys Ile Lys Thr Ile Val Gly Ile 885 890 895 900 gaa aaa atg tta tcttta tgt gga ggt gca gtc aaa ctt gga tta cca 3332 Glu Lys Met Leu Ser LeuCys Gly Gly Ala Val Lys Leu Gly Leu Pro 905 910 915 cag ata ctt tct aattta caa agt gct ttt caa aat gag cac tta agg 3380 Gln Ile Leu Ser Asn LeuGln Ser Ala Phe Gln Asn Glu His Leu Arg 920 925 930 ttt tat gca atc aaagct tgg ttc agt ttg ata tta gca acc aag gag 3428 Phe Tyr Ala Ile Lys AlaTrp Phe Ser Leu Ile Leu Ala Thr Lys Glu 935 940 945 ccc gag tat agt tcaatt gct ggt tta agt ctt gta att tta cct cct 3476 Pro Glu Tyr Ser Ser IleAla Gly Leu Ser Leu Val Ile Leu Pro Pro 950 955 960 tta ttc cct tat ttagaa cca caa gaa gca gag cta gta att caa ata 3524 Leu Phe Pro Tyr Leu GluPro Gln Glu Ala Glu Leu Val Ile Gln Ile 965 970 975 980 ttt gat ttt atttct tct gac aca cac aag tgc cta caa gga tta aag 3572 Phe Asp Phe Ile SerSer Asp Thr His Lys Cys Leu Gln Gly Leu Lys 985 990 995 tgg gct atc cccacc agt ctg gat tca gcg tgc ttt agc ctt aag 3617 Trp Ala Ile Pro Thr SerLeu Asp Ser Ala Cys Phe Ser Leu Lys 1000 1005 1010 gct aaa gaa ata ttctgt tcg ctt caa aat gaa gat ttt tac tct 3662 Ala Lys Glu Ile Phe Cys SerLeu Gln Asn Glu Asp Phe Tyr Ser 1015 1020 1025 gag ctt caa agt ata attaag tgt tta act aac gaa aat gag cca 3707 Glu Leu Gln Ser Ile Ile Lys CysLeu Thr Asn Glu Asn Glu Pro 1030 1035 1040 gtt tgt tat tta ggt tta caaaaa tta gaa ctt ttt ttt caa gcc 3752 Val Cys Tyr Leu Gly Leu Gln Lys LeuGlu Leu Phe Phe Gln Ala 1045 1050 1055 aag gtg gac gag tta cat gac acacta aat ttg gac ata tcc aac 3797 Lys Val Asp Glu Leu His Asp Thr Leu AsnLeu Asp Ile Ser Asn 1060 1065 1070 gaa gtt ctg gac caa tta cta aga tgcctt tta gat tgt tgt gta 3842 Glu Val Leu Asp Gln Leu Leu Arg Cys Leu LeuAsp Cys Cys Val 1075 1080 1085 aaa tat gct tca aca aat atg caa ata tcatat ctt gct gca aaa 3887 Lys Tyr Ala Ser Thr Asn Met Gln Ile Ser Tyr LeuAla Ala Lys 1090 1095 1100 aat ctt ggt gaa ttg ggt gcg ata gat ccc agccgc gcc aag gct 3932 Asn Leu Gly Glu Leu Gly Ala Ile Asp Pro Ser Arg AlaLys Ala 1105 1110 1115 caa cat att att aaa gaa aca gtt gtt ctt gat aacttt gaa aac 3977 Gln His Ile Ile Lys Glu Thr Val Val Leu Asp Asn Phe GluAsn 1120 1125 1130 gga gaa gaa agt ttg aag ttt att cta gat ttt atg caatcg cag 4022 Gly Glu Glu Ser Leu Lys Phe Ile Leu Asp Phe Met Gln Ser Gln1135 1140 1145 tta att cca gct ttc ctt gtt act act gat act aaa gca caaggt 4067 Leu Ile Pro Ala Phe Leu Val Thr Thr Asp Thr Lys Ala Gln Gly1150 1155 1160 ttt ctt gcc tat gct ctg caa gag ttt cta aag ctt ggt ggattc 4112 Phe Leu Ala Tyr Ala Leu Gln Glu Phe Leu Lys Leu Gly Gly Phe1165 1170 1175 aag tcc gca gtg att aat aaa aaa aag gga cta act gtg gtaaca 4157 Lys Ser Ala Val Ile Asn Lys Lys Lys Gly Leu Thr Val Val Thr1180 1185 1190 gaa cat tgg atg tct ttg cct gat tta tcc aaa cgt gtg cttata 4202 Glu His Trp Met Ser Leu Pro Asp Leu Ser Lys Arg Val Leu Ile1195 1200 1205 cca ttt tta act tcc aag tat cat tta aca cca atc ccc aaaatt 4247 Pro Phe Leu Thr Ser Lys Tyr His Leu Thr Pro Ile Pro Lys Ile1210 1215 1220 gac att cgg tac cct att tat aaa gaa aat gtt act att catact 4292 Asp Ile Arg Tyr Pro Ile Tyr Lys Glu Asn Val Thr Ile His Thr1225 1230 1235 tgg atg cag ttg ttt tct ctt aaa ttg atg gag tac gcc cattcg 4337 Trp Met Gln Leu Phe Ser Leu Lys Leu Met Glu Tyr Ala His Ser1240 1245 1250 caa aac gct gaa aaa ata ttt ggt att tgt tcg aaa gta gtgaaa 4382 Gln Asn Ala Glu Lys Ile Phe Gly Ile Cys Ser Lys Val Val Lys1255 1260 1265 gac caa gag gtt aac att ccc tgt ttt ctt ctt ccc ttt cttgtt 4427 Asp Gln Glu Val Asn Ile Pro Cys Phe Leu Leu Pro Phe Leu Val1270 1275 1280 tta aat gtt att tta acc gag tca gaa ctg gaa gtt aat aaagtc 4472 Leu Asn Val Ile Leu Thr Glu Ser Glu Leu Glu Val Asn Lys Val1285 1290 1295 att gaa gaa ttc cag ctt gtt att aat caa ccg gga cct gatgga 4517 Ile Glu Glu Phe Gln Leu Val Ile Asn Gln Pro Gly Pro Asp Gly1300 1305 1310 tta aat tcc gtg ggg caa caa aga tac acc tca ttt gta gatgta 4562 Leu Asn Ser Val Gly Gln Gln Arg Tyr Thr Ser Phe Val Asp Val1315 1320 1325 ttt ttt aag att gtg gat tac ctt aac aaa tgg ctt cgc atgcga 4607 Phe Phe Lys Ile Val Asp Tyr Leu Asn Lys Trp Leu Arg Met Arg1330 1335 1340 aag aag agg aat tgg gat aga cgt tct gcc att gca agg aaagag 4652 Lys Lys Arg Asn Trp Asp Arg Arg Ser Ala Ile Ala Arg Lys Glu1345 1350 1355 aac cgt tat atg tcg gtg gaa gat gct acc tct cga gaa tcatcg 4697 Asn Arg Tyr Met Ser Val Glu Asp Ala Thr Ser Arg Glu Ser Ser1360 1365 1370 atc tca aaa gtt gag tca ttt ctt tct cga ttt cct tca aaaaca 4742 Ile Ser Lys Val Glu Ser Phe Leu Ser Arg Phe Pro Ser Lys Thr1375 1380 1385 tta ggt att gtc tct tta aat tgt gga ttt cat gct cgt gcattg 4787 Leu Gly Ile Val Ser Leu Asn Cys Gly Phe His Ala Arg Ala Leu1390 1395 1400 ttt tat tgg gag caa cac ata cgt aat gct aca gct cca tatgca 4832 Phe Tyr Trp Glu Gln His Ile Arg Asn Ala Thr Ala Pro Tyr Ala1405 1410 1415 gct tta gag tcc gat tat aga gtt ttg cag gaa ata tat gctgga 4877 Ala Leu Glu Ser Asp Tyr Arg Val Leu Gln Glu Ile Tyr Ala Gly1420 1425 1430 att gat gat cca gat gaa atc gaa gca gtg tct tta aat ttccat 4922 Ile Asp Asp Pro Asp Glu Ile Glu Ala Val Ser Leu Asn Phe His1435 1440 1445 gat tac tcg ttt gat caa caa ctc ctt tta cat gaa aat tcagga 4967 Asp Tyr Ser Phe Asp Gln Gln Leu Leu Leu His Glu Asn Ser Gly1450 1455 1460 aca tgg gac tcg gct ttg agt tgt tac gaa att att att caaaag 5012 Thr Trp Asp Ser Ala Leu Ser Cys Tyr Glu Ile Ile Ile Gln Lys1465 1470 1475 gat cct gaa aat aaa aag gcg aaa atc ggt ttg ctt aac agcatg 5057 Asp Pro Glu Asn Lys Lys Ala Lys Ile Gly Leu Leu Asn Ser Met1480 1485 1490 ctg caa tcg ggg cat tat gaa tct ctt gtt ttg agt tta gattct 5102 Leu Gln Ser Gly His Tyr Glu Ser Leu Val Leu Ser Leu Asp Ser1495 1500 1505 ttt ata atc aat gac aac cac gag tat tcg aag atg tta aatttg 5147 Phe Ile Ile Asn Asp Asn His Glu Tyr Ser Lys Met Leu Asn Leu1510 1515 1520 ggt att gaa gct tca tgg cgt tcg cta tct att gat tcg ttaaaa 5192 Gly Ile Glu Ala Ser Trp Arg Ser Leu Ser Ile Asp Ser Leu Lys1525 1530 1535 aag tgt ctt tca aaa agc aac ttg gaa tct ttc gaa gct aaattg 5237 Lys Cys Leu Ser Lys Ser Asn Leu Glu Ser Phe Glu Ala Lys Leu1540 1545 1550 ggt agc ata ttt tac caa tac cta cgg aag gat tct ttt gctgaa 5282 Gly Ser Ile Phe Tyr Gln Tyr Leu Arg Lys Asp Ser Phe Ala Glu1555 1560 1565 ttg acg gag cgg ctg caa ccc ttg tac gtt gat gct gct acagca 5327 Leu Thr Glu Arg Leu Gln Pro Leu Tyr Val Asp Ala Ala Thr Ala1570 1575 1580 att gca aac aca ggc gcc cat tca gcc tat gat tgt tat gatatt 5372 Ile Ala Asn Thr Gly Ala His Ser Ala Tyr Asp Cys Tyr Asp Ile1585 1590 1595 tta tct aag ctg cac gca att aat gac ttt agt agg att gctgaa 5417 Leu Ser Lys Leu His Ala Ile Asn Asp Phe Ser Arg Ile Ala Glu1600 1605 1610 act gac gga att gtt tcc gac aat ctt gat att gtt ctt cgccgt 5462 Thr Asp Gly Ile Val Ser Asp Asn Leu Asp Ile Val Leu Arg Arg1615 1620 1625 cgg ctt agc caa gta gct ccg tac ggt aaa ttc aag cac caaatc 5507 Arg Leu Ser Gln Val Ala Pro Tyr Gly Lys Phe Lys His Gln Ile1630 1635 1640 ctg tcc act cac tta gtt ggc tat gaa aaa ttt gaa aac acgaag 5552 Leu Ser Thr His Leu Val Gly Tyr Glu Lys Phe Glu Asn Thr Lys1645 1650 1655 aaa act gct gaa ata tat ctc gag att gca aga ata tct cgaaaa 5597 Lys Thr Ala Glu Ile Tyr Leu Glu Ile Ala Arg Ile Ser Arg Lys1660 1665 1670 aat ggt caa ttt caa aga gcc ttc aat gcc atc ctc aaa gcaatg 5642 Asn Gly Gln Phe Gln Arg Ala Phe Asn Ala Ile Leu Lys Ala Met1675 1680 1685 gat tta gat aaa ccg cta gca aca ata gag cac gca caa tggtgg 5687 Asp Leu Asp Lys Pro Leu Ala Thr Ile Glu His Ala Gln Trp Trp1690 1695 1700 tgg cat caa ggg caa cat cgt aaa gct att tct gaa ttg aatttt 5732 Trp His Gln Gly Gln His Arg Lys Ala Ile Ser Glu Leu Asn Phe1705 1710 1715 tcg ctt aat aac aac atg ttt gat ttg gtt gat gag cat gaagaa 5777 Ser Leu Asn Asn Asn Met Phe Asp Leu Val Asp Glu His Glu Glu1720 1725 1730 aga cct aaa aat cgt aaa gaa act tta gga aat cca ctt aaagga 5822 Arg Pro Lys Asn Arg Lys Glu Thr Leu Gly Asn Pro Leu Lys Gly1735 1740 1745 aaa gtg ttc ttg aaa ctt aca aaa tgg ctc gga aaa gct ggccaa 5867 Lys Val Phe Leu Lys Leu Thr Lys Trp Leu Gly Lys Ala Gly Gln1750 1755 1760 ctg gga ttg aag gat ttg gag acg tat tat cat aaa gcg gtagag 5912 Leu Gly Leu Lys Asp Leu Glu Thr Tyr Tyr His Lys Ala Val Glu1765 1770 1775 att tac tca gaa tgt gag aat acg cat tat tat ctt ggc catcat 5957 Ile Tyr Ser Glu Cys Glu Asn Thr His Tyr Tyr Leu Gly His His1780 1785 1790 cga gtt tta atg tat gaa gaa gaa caa aag ctc cca gtt aatgaa 6002 Arg Val Leu Met Tyr Glu Glu Glu Gln Lys Leu Pro Val Asn Glu1795 1800 1805 cag agc gaa cga ttt tta agt ggt gag tta gta act cgc ataatt 6047 Gln Ser Glu Arg Phe Leu Ser Gly Glu Leu Val Thr Arg Ile Ile1810 1815 1820 aac gaa ttt ggt cga tct ttg tac tat ggt aca aat cat atatat 6092 Asn Glu Phe Gly Arg Ser Leu Tyr Tyr Gly Thr Asn His Ile Tyr1825 1830 1835 gaa agt atg cca aaa ttg ctc aca ctg tgg ctt gat ttt ggggcc 6137 Glu Ser Met Pro Lys Leu Leu Thr Leu Trp Leu Asp Phe Gly Ala1840 1845 1850 gaa gaa ctt cgc tta tct aaa gat gac ggc gaa aag tac tttcgt 6182 Glu Glu Leu Arg Leu Ser Lys Asp Asp Gly Glu Lys Tyr Phe Arg1855 1860 1865 gaa cac att atc tct tcg aga aaa aaa tct ttg gaa ctt atgaat 6227 Glu His Ile Ile Ser Ser Arg Lys Lys Ser Leu Glu Leu Met Asn1870 1875 1880 tcg aat gtt tgt cgc ctt tct atg aaa att cct caa tac tttttt 6272 Ser Asn Val Cys Arg Leu Ser Met Lys Ile Pro Gln Tyr Phe Phe1885 1890 1895 ctg gtt gca tta tcc caa atg ata tcc aga gta tgc cat ccaaat 6317 Leu Val Ala Leu Ser Gln Met Ile Ser Arg Val Cys His Pro Asn1900 1905 1910 aat aaa gtt tat aaa att ttg gaa cat ata att gca aac gttgta 6362 Asn Lys Val Tyr Lys Ile Leu Glu His Ile Ile Ala Asn Val Val1915 1920 1925 gca tct tat cct ggg gag acg cta tgg caa tta atg gca acaata 6407 Ala Ser Tyr Pro Gly Glu Thr Leu Trp Gln Leu Met Ala Thr Ile1930 1935 1940 aaa tcg act tct caa aag cgc tcg ctt cgt gga aaa agc atttta 6452 Lys Ser Thr Ser Gln Lys Arg Ser Leu Arg Gly Lys Ser Ile Leu1945 1950 1955 aat gtt tta cat tct agg aag ctt tct atg tct tcc aaa gttgat 6497 Asn Val Leu His Ser Arg Lys Leu Ser Met Ser Ser Lys Val Asp1960 1965 1970 ata aaa gca ctc agt caa tct gca att ctc att act gaa aagtta 6542 Ile Lys Ala Leu Ser Gln Ser Ala Ile Leu Ile Thr Glu Lys Leu1975 1980 1985 atc aat ttg tgc aat aca agg att aac agt aaa tct gta aaaatg 6587 Ile Asn Leu Cys Asn Thr Arg Ile Asn Ser Lys Ser Val Lys Met1990 1995 2000 agc tta aag gat cat ttt cgg ctt tct ttt gat gat ccg gtagat 6632 Ser Leu Lys Asp His Phe Arg Leu Ser Phe Asp Asp Pro Val Asp2005 2010 2015 tta gtc att cct gct aaa tca ttt tta gac att act tta ccagct 6677 Leu Val Ile Pro Ala Lys Ser Phe Leu Asp Ile Thr Leu Pro Ala2020 2025 2030 aaa gat gct aac aga gct agt cat tat cca ttt cca aaa actcag 6722 Lys Asp Ala Asn Arg Ala Ser His Tyr Pro Phe Pro Lys Thr Gln2035 2040 2045 cct act ctg ttg aaa ttt gag gat gag gtg gat ata atg aactct 6767 Pro Thr Leu Leu Lys Phe Glu Asp Glu Val Asp Ile Met Asn Ser2050 2055 2060 ctt caa aaa cca aga aaa gtg tac gtt aga ggt acg gat ggcaac 6812 Leu Gln Lys Pro Arg Lys Val Tyr Val Arg Gly Thr Asp Gly Asn2065 2070 2075 tta tac cca ttc ttg tgc aaa ccc aaa gat gat ctt cgt aaggat 6857 Leu Tyr Pro Phe Leu Cys Lys Pro Lys Asp Asp Leu Arg Lys Asp2080 2085 2090 gct aga ttg atg gaa ttt aat aat ctt att tgt aaa ata ttgagg 6902 Ala Arg Leu Met Glu Phe Asn Asn Leu Ile Cys Lys Ile Leu Arg2095 2100 2105 aaa gat caa gaa gcg aac aga agg aac ttg tgt att aga acttat 6947 Lys Asp Gln Glu Ala Asn Arg Arg Asn Leu Cys Ile Arg Thr Tyr2110 2115 2120 gtt gtt att cct tta aat gaa gaa tgc gga ttt atc gaa tgggta 6992 Val Val Ile Pro Leu Asn Glu Glu Cys Gly Phe Ile Glu Trp Val2125 2130 2135 aat cat act cgt cca ttt aga gaa att ttg tta aaa agc tataga 7037 Asn His Thr Arg Pro Phe Arg Glu Ile Leu Leu Lys Ser Tyr Arg2140 2145 2150 cag aaa aac att ccc ata tca tat caa gaa atc aaa gtt gattta 7082 Gln Lys Asn Ile Pro Ile Ser Tyr Gln Glu Ile Lys Val Asp Leu2155 2160 2165 gac ttt gca ctg cga agt cct aac cct ggt gat ata ttt gaaaag 7127 Asp Phe Ala Leu Arg Ser Pro Asn Pro Gly Asp Ile Phe Glu Lys2170 2175 2180 aaa atc tta ccg aaa ttt cct cca gtt ttt tat gag tgg tttgtt 7172 Lys Ile Leu Pro Lys Phe Pro Pro Val Phe Tyr Glu Trp Phe Val2185 2190 2195 gaa tct ttc cca gaa cca aat aat tgg gtt act agt aga caaaac 7217 Glu Ser Phe Pro Glu Pro Asn Asn Trp Val Thr Ser Arg Gln Asn2200 2205 2210 tat tgc cga act tta gca gta atg tca ata gtt ggc tac gttttg 7262 Tyr Cys Arg Thr Leu Ala Val Met Ser Ile Val Gly Tyr Val Leu2215 2220 2225 ggt ttg gga gat cgc cat ggc gaa aac ata ttg ttt gat gaattt 7307 Gly Leu Gly Asp Arg His Gly Glu Asn Ile Leu Phe Asp Glu Phe2230 2235 2240 aca ggt gaa gct atc cat gtc gat ttc aac tgt ctt ttt gataaa 7352 Thr Gly Glu Ala Ile His Val Asp Phe Asn Cys Leu Phe Asp Lys2245 2250 2255 ggt ctt act ttt gaa aaa cct gaa aag gtg ccg ttc aga ttaact 7397 Gly Leu Thr Phe Glu Lys Pro Glu Lys Val Pro Phe Arg Leu Thr2260 2265 2270 cat aat atg gta gat gca atg ggt ccg aca ggt tat gaa gggggt 7442 His Asn Met Val Asp Ala Met Gly Pro Thr Gly Tyr Glu Gly Gly2275 2280 2285 ttc agg aaa gct agc gaa ata acg atg cgg ctt ctt cgc tcaaac 7487 Phe Arg Lys Ala Ser Glu Ile Thr Met Arg Leu Leu Arg Ser Asn2290 2295 2300 caa gat aca ttg atg agc gta cta gag tct ttc cta cat gatcct 7532 Gln Asp Thr Leu Met Ser Val Leu Glu Ser Phe Leu His Asp Pro2305 2310 2315 tta gtc gag tgg aat aga aag aag tcg tca agc aag tac ccgaat 7577 Leu Val Glu Trp Asn Arg Lys Lys Ser Ser Ser Lys Tyr Pro Asn2320 2325 2330 aat gaa gca aat gaa gtt ttg gat ata att cgc aaa aaa tttcaa 7622 Asn Glu Ala Asn Glu Val Leu Asp Ile Ile Arg Lys Lys Phe Gln2335 2340 2345 ggc ttt atg cca ggg gag acg ata cct tta tct att gaa gggcaa 7667 Gly Phe Met Pro Gly Glu Thr Ile Pro Leu Ser Ile Glu Gly Gln2350 2355 2360 att caa gaa ttg atc aaa tct gct gtc aac cca aaa aac ctggta 7712 Ile Gln Glu Leu Ile Lys Ser Ala Val Asn Pro Lys Asn Leu Val2365 2370 2375 gaa atg tac att ggt tgg gct gct tat ttc tagcattttactaacaaaaa 7762 Glu Met Tyr Ile Gly Trp Ala Ala Tyr Phe 2380 2385tttcaatgaa caagctaccc attattaaac ttatgatttg aatcgaagat attttattta 7822ttaatccgat gaagaattct cgctgagttg ttcaatttct tgtaattttc cttccatttc 7882taaatcgtcg attcgcttaa atagggcact ggctttttgt gcatttttct ctcgtaaagc 7942agcttctgat tgaaaaaaag ctatatctgt ttctgagtca tcatccgaat caacaatata 8002ttttgcagat cgacctgcag 8022 4 2386 PRT Homo sapiens 4 Met Ser Gln His AlaLys Arg Lys Ala Gly Ser Leu Asp Leu Ser Pro 1 5 10 15 Arg Gly Leu AspAsp Arg Gln Ala Phe Gly Gln Leu Leu Lys Glu Val 20 25 30 Leu Ala Leu AspLys Glu His Glu Leu Gly Arg Ser Asn Ser Leu Pro 35 40 45 Ser Met Thr SerGlu Leu Val Glu Val Leu Ile Glu Val Gly Leu Leu 50 55 60 Ala Phe Lys HisAsp Asp Ser Lys Ser Glu Phe Ile Ser Pro Lys Met 65 70 75 80 Leu Lys GluAla His Leu Ser Leu Gln Ala Leu Met Leu Ile Leu Lys 85 90 95 Arg Ser ProThr Val Leu Arg Glu Ile Lys Ser Ser Val Thr Leu Leu 100 105 110 Asp TrpIle Leu Pro Arg Thr Ile Ser Leu Phe Ala Asp Ile Arg Phe 115 120 125 IleLys Leu Phe Asp Ser Leu Lys Glu Phe His Lys Leu Ile Tyr Gln 130 135 140Leu Ile Ser Glu Lys Ser Phe Leu Trp Asp Leu Tyr Ala Ser Phe Met 145 150155 160 Arg Tyr Trp Lys Tyr Tyr Ile Thr Asn Val Ser Ser Ile Val Leu Gln165 170 175 Ile Thr Asn Ala Thr Phe Pro Tyr Lys Met Pro Ser Pro Asn SerGln 180 185 190 Pro Leu Gln Ser Ile Ser Pro Asn Tyr Pro Thr His Arg GluAsp Lys 195 200 205 Phe Asp Leu Leu Ile Ile Asn Ile Glu Glu Ala Cys ThrPhe Phe Phe 210 215 220 Glu Ser Ala His Phe Phe Ala Gln Cys Ser Tyr LeuLys Lys Ser Asn 225 230 235 240 Phe Pro Ser Pro Pro Leu Phe Thr Ala TrpThr Trp Ile Lys Pro Cys 245 250 255 Phe Phe Asn Phe Val Ile Leu Leu LysArg Ile Ser Ile Gly Asp Ser 260 265 270 Gln Leu Phe Leu His Leu His SerArg Ile Val Gln Thr Leu Cys Cys 275 280 285 Phe Ser Leu Asn Phe Ile TyrHis Gly Leu Pro Ile Cys Glu Lys Ser 290 295 300 Lys His Ile Leu Met SerSer Ile Asn Leu Thr Leu Gly Ser Leu Lys 305 310 315 320 Lys Thr Tyr ThrVal Ala Asn Thr Ala Ile Ser Leu Phe Phe Leu Ser 325 330 335 Leu Phe ValLeu Pro Lys Thr Val Ala Gly Leu Phe Tyr Pro Phe Gly 340 345 350 Val SerLeu Leu Ser Asp Phe Lys Val Leu Glu Gln Leu Glu Pro Asp 355 360 365 SerAsp Leu Lys Lys Ala Ile Ile Leu Phe Lys Cys Arg Tyr Gln Ser 370 375 380Ser Glu Ile Asp Gln Thr Thr Leu Arg Ala Phe Gly Glu Ile Cys Thr 385 390395 400 Gly Lys Leu Glu Asn Thr Leu Phe Ser Asn Ser Glu Leu Asn Leu Phe405 410 415 Leu Leu His Tyr Leu Ser Leu Asp Asn Asp Leu Ser Asn Ile LeuLys 420 425 430 Val Asp Phe Gln Asn Gly His Asn Ile Cys Thr Phe Ala LysTrp Cys 435 440 445 Ile Asn Asn Asn Leu Asp Glu Pro Ser Asn Leu Lys HisPhe Arg Glu 450 455 460 Met Leu Asp Tyr Tyr Ser Ser His Asn Val Thr IleSer Glu Asp Asp 465 470 475 480 Leu Lys Asn Phe Ser Leu Val Leu Cys ThrHis Val Ala Lys Val Asn 485 490 495 Glu Lys Thr Asn Ser Ile Phe Arg ThrTyr Glu Val His Gly Cys Glu 500 505 510 Val Cys Asn Ser Phe Cys Leu LeuPhe Asp Glu Arg Ser Pro Phe Lys 515 520 525 Ile Pro Tyr His Glu Leu PheCys Ala Leu Leu Lys Asn Pro Asp Ile 530 535 540 Ile Ser Ser Ser Val LysGln Ser Leu Leu Leu Asp Gly Phe Phe Arg 545 550 555 560 Trp Ser Gln HisCys Ser Asn Phe Asn Lys Glu Ser Met Leu Ser Leu 565 570 575 Arg Glu PheIle Met Lys Ala Leu Ala Ser Thr Ser Arg Cys Leu Arg 580 585 590 Val ValAla Ala Lys Val Leu Pro Ile Phe Ile Lys Gly Pro Asn Asn 595 600 605 LeuAsp Ile Val Glu Phe His Lys Glu Ser Lys Ala Leu Ile Phe Asn 610 615 620Thr Leu Lys Ile Leu Ala Val Glu Asn Thr Ala Ile Leu Glu Thr Val 625 630635 640 Ile Leu Ser Trp Ile Ser Leu Ser Arg Val Val Glu Glu Glu Glu Leu645 650 655 His Phe Val Leu Leu Glu Val Ile Ser Ser Val Ile Asn Ser GlyIle 660 665 670 Phe Tyr Gln Gly Ile Gly Leu Ser Ala Leu Gln Gln Ile AlaSer Thr 675 680 685 Arg His Ile Ser Val Trp Gln Leu Leu Ser Pro Tyr TrpPro Thr Val 690 695 700 Ser Val Ala Ile Val Gln Gly Met Gly Lys Lys ProAsn Ile Ala Ser 705 710 715 720 Leu Phe Ala Gln Leu Met Asn Ile Ser GluGly Asp Phe Leu Ile Arg 725 730 735 Thr Gln Ala Tyr Thr Leu Pro Phe LeuVal Leu Thr Lys Asn Lys Ala 740 745 750 Leu Ile Val Arg Ile Ala Glu LeuSer Gln Ser Asp Val Ala Thr Leu 755 760 765 Cys Leu Thr Asn Met His LysIle Leu Ala Ser Leu Leu Thr Thr Asp 770 775 780 His Pro Asn Leu Glu GluSer Val Met Leu Leu Leu Ser Leu Ala Thr 785 790 795 800 Ser Asp Phe GluLys Val Asp Leu Thr Ser Leu Leu Arg Ser Asp Pro 805 810 815 Ile Ser IleThr Val Glu Leu Leu Gln Leu Tyr Gln Asn Asp Val Pro 820 825 830 His GluLys Ile Glu Asn Ala Leu Arg Lys Val Ala Met Ile Val Ser 835 840 845 GlnVal Val Asn Asp Glu Asp Leu Ser Asn Lys Glu Leu Leu Tyr Asp 850 855 860Phe Phe Asn Asn His Ile Leu Gly Ile Leu Ala Glu Phe Ser Asn Ile 865 870875 880 Leu Asn Asp Leu Lys Gly Lys Thr Ser Ile Asn Glu Lys Ile Lys Thr885 890 895 Ile Val Gly Ile Glu Lys Met Leu Ser Leu Cys Gly Gly Ala ValLys 900 905 910 Leu Gly Leu Pro Gln Ile Leu Ser Asn Leu Gln Ser Ala PheGln Asn 915 920 925 Glu His Leu Arg Phe Tyr Ala Ile Lys Ala Trp Phe SerLeu Ile Leu 930 935 940 Ala Thr Lys Glu Pro Glu Tyr Ser Ser Ile Ala GlyLeu Ser Leu Val 945 950 955 960 Ile Leu Pro Pro Leu Phe Pro Tyr Leu GluPro Gln Glu Ala Glu Leu 965 970 975 Val Ile Gln Ile Phe Asp Phe Ile SerSer Asp Thr His Lys Cys Leu 980 985 990 Gln Gly Leu Lys Trp Ala Ile ProThr Ser Leu Asp Ser Ala Cys Phe 995 1000 1005 Ser Leu Lys Ala Lys GluIle Phe Cys Ser Leu Gln Asn Glu Asp 1010 1015 1020 Phe Tyr Ser Glu LeuGln Ser Ile Ile Lys Cys Leu Thr Asn Glu 1025 1030 1035 Asn Glu Pro ValCys Tyr Leu Gly Leu Gln Lys Leu Glu Leu Phe 1040 1045 1050 Phe Gln AlaLys Val Asp Glu Leu His Asp Thr Leu Asn Leu Asp 1055 1060 1065 Ile SerAsn Glu Val Leu Asp Gln Leu Leu Arg Cys Leu Leu Asp 1070 1075 1080 CysCys Val Lys Tyr Ala Ser Thr Asn Met Gln Ile Ser Tyr Leu 1085 1090 1095Ala Ala Lys Asn Leu Gly Glu Leu Gly Ala Ile Asp Pro Ser Arg 1100 11051110 Ala Lys Ala Gln His Ile Ile Lys Glu Thr Val Val Leu Asp Asn 11151120 1125 Phe Glu Asn Gly Glu Glu Ser Leu Lys Phe Ile Leu Asp Phe Met1130 1135 1140 Gln Ser Gln Leu Ile Pro Ala Phe Leu Val Thr Thr Asp ThrLys 1145 1150 1155 Ala Gln Gly Phe Leu Ala Tyr Ala Leu Gln Glu Phe LeuLys Leu 1160 1165 1170 Gly Gly Phe Lys Ser Ala Val Ile Asn Lys Lys LysGly Leu Thr 1175 1180 1185 Val Val Thr Glu His Trp Met Ser Leu Pro AspLeu Ser Lys Arg 1190 1195 1200 Val Leu Ile Pro Phe Leu Thr Ser Lys TyrHis Leu Thr Pro Ile 1205 1210 1215 Pro Lys Ile Asp Ile Arg Tyr Pro IleTyr Lys Glu Asn Val Thr 1220 1225 1230 Ile His Thr Trp Met Gln Leu PheSer Leu Lys Leu Met Glu Tyr 1235 1240 1245 Ala His Ser Gln Asn Ala GluLys Ile Phe Gly Ile Cys Ser Lys 1250 1255 1260 Val Val Lys Asp Gln GluVal Asn Ile Pro Cys Phe Leu Leu Pro 1265 1270 1275 Phe Leu Val Leu AsnVal Ile Leu Thr Glu Ser Glu Leu Glu Val 1280 1285 1290 Asn Lys Val IleGlu Glu Phe Gln Leu Val Ile Asn Gln Pro Gly 1295 1300 1305 Pro Asp GlyLeu Asn Ser Val Gly Gln Gln Arg Tyr Thr Ser Phe 1310 1315 1320 Val AspVal Phe Phe Lys Ile Val Asp Tyr Leu Asn Lys Trp Leu 1325 1330 1335 ArgMet Arg Lys Lys Arg Asn Trp Asp Arg Arg Ser Ala Ile Ala 1340 1345 1350Arg Lys Glu Asn Arg Tyr Met Ser Val Glu Asp Ala Thr Ser Arg 1355 13601365 Glu Ser Ser Ile Ser Lys Val Glu Ser Phe Leu Ser Arg Phe Pro 13701375 1380 Ser Lys Thr Leu Gly Ile Val Ser Leu Asn Cys Gly Phe His Ala1385 1390 1395 Arg Ala Leu Phe Tyr Trp Glu Gln His Ile Arg Asn Ala ThrAla 1400 1405 1410 Pro Tyr Ala Ala Leu Glu Ser Asp Tyr Arg Val Leu GlnGlu Ile 1415 1420 1425 Tyr Ala Gly Ile Asp Asp Pro Asp Glu Ile Glu AlaVal Ser Leu 1430 1435 1440 Asn Phe His Asp Tyr Ser Phe Asp Gln Gln LeuLeu Leu His Glu 1445 1450 1455 Asn Ser Gly Thr Trp Asp Ser Ala Leu SerCys Tyr Glu Ile Ile 1460 1465 1470 Ile Gln Lys Asp Pro Glu Asn Lys LysAla Lys Ile Gly Leu Leu 1475 1480 1485 Asn Ser Met Leu Gln Ser Gly HisTyr Glu Ser Leu Val Leu Ser 1490 1495 1500 Leu Asp Ser Phe Ile Ile AsnAsp Asn His Glu Tyr Ser Lys Met 1505 1510 1515 Leu Asn Leu Gly Ile GluAla Ser Trp Arg Ser Leu Ser Ile Asp 1520 1525 1530 Ser Leu Lys Lys CysLeu Ser Lys Ser Asn Leu Glu Ser Phe Glu 1535 1540 1545 Ala Lys Leu GlySer Ile Phe Tyr Gln Tyr Leu Arg Lys Asp Ser 1550 1555 1560 Phe Ala GluLeu Thr Glu Arg Leu Gln Pro Leu Tyr Val Asp Ala 1565 1570 1575 Ala ThrAla Ile Ala Asn Thr Gly Ala His Ser Ala Tyr Asp Cys 1580 1585 1590 TyrAsp Ile Leu Ser Lys Leu His Ala Ile Asn Asp Phe Ser Arg 1595 1600 1605Ile Ala Glu Thr Asp Gly Ile Val Ser Asp Asn Leu Asp Ile Val 1610 16151620 Leu Arg Arg Arg Leu Ser Gln Val Ala Pro Tyr Gly Lys Phe Lys 16251630 1635 His Gln Ile Leu Ser Thr His Leu Val Gly Tyr Glu Lys Phe Glu1640 1645 1650 Asn Thr Lys Lys Thr Ala Glu Ile Tyr Leu Glu Ile Ala ArgIle 1655 1660 1665 Ser Arg Lys Asn Gly Gln Phe Gln Arg Ala Phe Asn AlaIle Leu 1670 1675 1680 Lys Ala Met Asp Leu Asp Lys Pro Leu Ala Thr IleGlu His Ala 1685 1690 1695 Gln Trp Trp Trp His Gln Gly Gln His Arg LysAla Ile Ser Glu 1700 1705 1710 Leu Asn Phe Ser Leu Asn Asn Asn Met PheAsp Leu Val Asp Glu 1715 1720 1725 His Glu Glu Arg Pro Lys Asn Arg LysGlu Thr Leu Gly Asn Pro 1730 1735 1740 Leu Lys Gly Lys Val Phe Leu LysLeu Thr Lys Trp Leu Gly Lys 1745 1750 1755 Ala Gly Gln Leu Gly Leu LysAsp Leu Glu Thr Tyr Tyr His Lys 1760 1765 1770 Ala Val Glu Ile Tyr SerGlu Cys Glu Asn Thr His Tyr Tyr Leu 1775 1780 1785 Gly His His Arg ValLeu Met Tyr Glu Glu Glu Gln Lys Leu Pro 1790 1795 1800 Val Asn Glu GlnSer Glu Arg Phe Leu Ser Gly Glu Leu Val Thr 1805 1810 1815 Arg Ile IleAsn Glu Phe Gly Arg Ser Leu Tyr Tyr Gly Thr Asn 1820 1825 1830 His IleTyr Glu Ser Met Pro Lys Leu Leu Thr Leu Trp Leu Asp 1835 1840 1845 PheGly Ala Glu Glu Leu Arg Leu Ser Lys Asp Asp Gly Glu Lys 1850 1855 1860Tyr Phe Arg Glu His Ile Ile Ser Ser Arg Lys Lys Ser Leu Glu 1865 18701875 Leu Met Asn Ser Asn Val Cys Arg Leu Ser Met Lys Ile Pro Gln 18801885 1890 Tyr Phe Phe Leu Val Ala Leu Ser Gln Met Ile Ser Arg Val Cys1895 1900 1905 His Pro Asn Asn Lys Val Tyr Lys Ile Leu Glu His Ile IleAla 1910 1915 1920 Asn Val Val Ala Ser Tyr Pro Gly Glu Thr Leu Trp GlnLeu Met 1925 1930 1935 Ala Thr Ile Lys Ser Thr Ser Gln Lys Arg Ser LeuArg Gly Lys 1940 1945 1950 Ser Ile Leu Asn Val Leu His Ser Arg Lys LeuSer Met Ser Ser 1955 1960 1965 Lys Val Asp Ile Lys Ala Leu Ser Gln SerAla Ile Leu Ile Thr 1970 1975 1980 Glu Lys Leu Ile Asn Leu Cys Asn ThrArg Ile Asn Ser Lys Ser 1985 1990 1995 Val Lys Met Ser Leu Lys Asp HisPhe Arg Leu Ser Phe Asp Asp 2000 2005 2010 Pro Val Asp Leu Val Ile ProAla Lys Ser Phe Leu Asp Ile Thr 2015 2020 2025 Leu Pro Ala Lys Asp AlaAsn Arg Ala Ser His Tyr Pro Phe Pro 2030 2035 2040 Lys Thr Gln Pro ThrLeu Leu Lys Phe Glu Asp Glu Val Asp Ile 2045 2050 2055 Met Asn Ser LeuGln Lys Pro Arg Lys Val Tyr Val Arg Gly Thr 2060 2065 2070 Asp Gly AsnLeu Tyr Pro Phe Leu Cys Lys Pro Lys Asp Asp Leu 2075 2080 2085 Arg LysAsp Ala Arg Leu Met Glu Phe Asn Asn Leu Ile Cys Lys 2090 2095 2100 IleLeu Arg Lys Asp Gln Glu Ala Asn Arg Arg Asn Leu Cys Ile 2105 2110 2115Arg Thr Tyr Val Val Ile Pro Leu Asn Glu Glu Cys Gly Phe Ile 2120 21252130 Glu Trp Val Asn His Thr Arg Pro Phe Arg Glu Ile Leu Leu Lys 21352140 2145 Ser Tyr Arg Gln Lys Asn Ile Pro Ile Ser Tyr Gln Glu Ile Lys2150 2155 2160 Val Asp Leu Asp Phe Ala Leu Arg Ser Pro Asn Pro Gly AspIle 2165 2170 2175 Phe Glu Lys Lys Ile Leu Pro Lys Phe Pro Pro Val PheTyr Glu 2180 2185 2190 Trp Phe Val Glu Ser Phe Pro Glu Pro Asn Asn TrpVal Thr Ser 2195 2200 2205 Arg Gln Asn Tyr Cys Arg Thr Leu Ala Val MetSer Ile Val Gly 2210 2215 2220 Tyr Val Leu Gly Leu Gly Asp Arg His GlyGlu Asn Ile Leu Phe 2225 2230 2235 Asp Glu Phe Thr Gly Glu Ala Ile HisVal Asp Phe Asn Cys Leu 2240 2245 2250 Phe Asp Lys Gly Leu Thr Phe GluLys Pro Glu Lys Val Pro Phe 2255 2260 2265 Arg Leu Thr His Asn Met ValAsp Ala Met Gly Pro Thr Gly Tyr 2270 2275 2280 Glu Gly Gly Phe Arg LysAla Ser Glu Ile Thr Met Arg Leu Leu 2285 2290 2295 Arg Ser Asn Gln AspThr Leu Met Ser Val Leu Glu Ser Phe Leu 2300 2305 2310 His Asp Pro LeuVal Glu Trp Asn Arg Lys Lys Ser Ser Ser Lys 2315 2320 2325 Tyr Pro AsnAsn Glu Ala Asn Glu Val Leu Asp Ile Ile Arg Lys 2330 2335 2340 Lys PheGln Gly Phe Met Pro Gly Glu Thr Ile Pro Leu Ser Ile 2345 2350 2355 GluGly Gln Ile Gln Glu Leu Ile Lys Ser Ala Val Asn Pro Lys 2360 2365 2370Asn Leu Val Glu Met Tyr Ile Gly Trp Ala Ala Tyr Phe 2375 2380 2385 5 30DNA Artificial sequence Primer 5 gttttcgcca tggcgcgctc ccaaacccaa 30 630 DNA Artificial sequence Primer 6 ttcatcaaac aatatctttt cgccatggcg 307 30 DNA Artificial sequence Primer 7 caaaaagaca gttgaattcg acatggatag30 8 33 DNA Artificial sequence Primer 8 gacgcagaat tcaccagtcaaagaatcaaa gag 33 9 20 DNA Artificial sequence Primer 9 tggatgatgacagctgtgtc 20 10 20 DNA Artificial sequence Primer 10 tgtagtcgctgctcaatgtc 20 11 23 DNA Artificial sequence Primer 11 tggtttctgagaacattccc tga 23 12 10 PRT Artificial sequence Rad3/Esrlp 12 Lys PhePro Pro Xaa Xaa Tyr Xaa Trp Phe 1 5 10 13 7 PRT Artificial sequenceoligonucleutide oDH18 13 Leu Gly Leu Gly Asp Arg His 1 5 14 6 PRTArtificial sequence oligonucleotide oDH-16 14 His Val Asp Phe Xaa Cys 15

1. A polynucleotide in substantially isolated form capable ofhybridising selectively to Seq.ID No. 1 or to its complement.
 2. Apolynucleotide according to claim 1 which comprises of Seq.ID No. 1 or afragment thereof.
 3. A polynucleotide probe which comprises a fragmentof at least 15 nucleotides of a polynucleotide as defined in claim 1 or2.
 4. A polynucleotide in substantially isolated form comprising Seq.IDNo. 3 or its complement.
 5. A polypeptide in substantially isolated formwhich comprises either of the sequences set out in Seq ID Nos. 2 or 4polypeptide substantially homologous thereto, or a fragment of thepolypeptide of Seq. ID No.
 2. 6. A polynucleotide in substantiallyisolated form encoding a polypeptide according to claim
 5. 7. A vectorcarrying a polynucleotide as defined in claim 1, 2 or
 6. 8. An antibodycapable of binding the polypeptide of Seq. ID. No. 2 or fragmentthereof.
 9. A method for detecting the presence or absence of apolynucleotide as defined in claim 1 in a human or animal body samplewhich comprises: bringing a human or animal body sample containing DNAor RNA into contact with a probe comprising a polynucleotide or primeras defined in claim 1 under hybridizing conditions; and detecting anyduplex formed between the probe and nuclei acid in the sample.
 10. Amethod of detecting polypeptides as defined in claim 6 present inbiological samples which comprises: (a) providing an antibody accordingto claim 7; (b) incubating a biological sample with said antibody underconditions which allow for the formation of an antibody-antigen complex;and (c) determining whether antibody-antigen complex comprising saidantibody is formed.
 11. An assay method for screening candidatesubstances for anti-cancer therapy which comprises: (a) providing apolypeptide of the invention which retains lipid kinase activity and asubstrate for said kinase, under conditions and with reagents such thatthe kinase activity will act upon the substrate; (b) bringing saidpolypeptide and substrate into contact with a candidate substance; (c)measuring the degree of decrease in the kinase activity of thepolypeptide; and (d) selecting a candidate substance which provides adecrease in activity.
 12. An assay method for screening candidatesubstances for anti-cancer therapy which comprises: (a) (i) incubating apolypeptide of the invention with another as polypeptide of theinvention, which may be the same or different to the first polypeptide,under conditions which allow the first polypeptide to bind to the secondpolypeptide to form a complex; (ii) bringing the complex thus formedinto contact with a candidate substance; or (a) incubating a polypeptideof the invention with another polypeptide of the invention, which may bethe same as or different to the first polypeptide, under conditionswhich allow the first polypeptide to bind to the second polypeptide toform a complex and in the presence of a candidate substance; and (b)determining whether the candidate substance inhibits binding of thefirst polypeptide to the second polypeptide and (c) selecting acandidate substance which inhibits binding of the first polypeptide tothe second polypeptide.
 13. A method according to claim 12 wherein saidfirst polypeptide can be distinguished from said second polypeptide. 14.A method of treating cancer in a patient which comprises administeringto said patient a therapeutically effective amount of a candidatesubstance selected according to the method of any one of claims 11 to13.
 15. A method of increasing the susceptibility of cancer cells in apatient to chemotherapy and/or radiotherapy which comprisesadministering to said patient a therapeutically effective amount of acandidate substance selected according to the method of any one ofclaims 11 to
 13. 16. Use of a candidate substance selected according tothe method of any one of claims 11 to 13 for the treatment of cancer.17. Use of a candidate substance selected according to the method of anyone of claims 11 to 13 for increasing the susceptibility of cancer cellsto chemotherapy and/or radiotherapy.